Inhibitors of IAP

ABSTRACT

The invention provides novel inhibitors of IAP that are useful as therapeutic agents for treating malignancies where the compounds have the general formula I: 
                         
wherein X, Y, A, R 1 , R 2 , R 3 , R 4 , R 4 ′, R 5 , R 5 ′, R 6  and R 6 ′ are as described herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/739,030, filed on 23 Apr. 2007 now abandoned, which is a continuationof U.S. patent application Ser. No. 11/174,784, filed on 5 Jul. 2005,now U.S. Pat. No. 7,224,851, which claims priority under 35 USC §119(e)to U.S. provisional application Ser. No. 60/585,501, filed 2 Jul. 2004,the entire disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to organic compounds useful for therapyand/or prophylaxis in a mammal, and in particular to inhibitors of IAPproteins useful for treating cancers.

BACKGROUND OF THE INVENTION

Apoptosis or programmed cell death is a genetically and biochemicallyregulated mechanism that plays an important role in development andhomeostasis in invertebrates as well as vertebrates. Aberrancies inapoptosis that lead to premature cell death have been linked to avariety of developmental disorders. Deficiencies in apoptosis thatresult in the lack of cell death have been linked to cancer and chronicviral infections (Thompson et al., (1995) Science 267, 1456-1462).

One of the key effector molecules in apoptosis are the caspases(cysteine containing aspartate specific proteases). Caspases are strongproteases, cleaving after aspartic acid residues and once activated,digest vital cell proteins from within the cell. Since caspases are suchstrong proteases, tight control of this family of proteins is necessaryto prevent premature cell death. In general, caspases are synthesized aslargely inactive zymogens that require proteolytic processing in orderto be active. This proteolytic processing is only one of the ways inwhich caspases are regulated. The second mechanism is through a familyof proteins that bind and inhibit caspases.

A family of molecules that inhibit caspases are the Inhibitors ofApoptosis (IAP) (Deveraux et al., J Clin Immunol (1999), 19:388-398).IAPs were originally discovered in baculovirus by their functionalability to substitute for P35 protein, an anti-apoptotic gene (Crook etal. (1993) J Virology 67, 2168-2174). IAPs have been described inorganisms ranging from Drosophila to human. Regardless of their origin,structurally, IAPs comprise one to three Baculovirus IAP repeat (BIR)domains, and most of them also possess a carboxyl-terminal RING fingermotif. The BIR domain itself is a zinc binding domain of about 70residues comprising 4 alpha-helices and 3 beta strands, with cysteineand histidine residues that coordinate the zinc ion (Hinds et al.,(1999) Nat. Struct. Biol. 6, 648-651). It is the BIR domain that isbelieved to cause the anti-apoptotic effect by inhibiting the caspasesand thus inhibiting apoptosis. As an example, human X-chromosome linkedIAP (XIAP) inhibits caspase 3, caspase 7 and the Apaf-1-cytochrome Cmediated activation of caspase 9 (Deveraux et al., (1998) EMBO J. 17,2215-2223). Caspases 3 and 7 are inhibited by the BIR2 domain of XIAP,while the BIR3 domain of XIAP is responsible for the inhibition ofcaspase 9 activity. XIAP is expressed ubiquitously in most adult andfetal tissues (Liston et al, Nature, 1996, 379(6563):349), and isoverexpressed in a number of tumor cell lines of the NCI 60 cell linepanel (Fong et al, Genomics, 2000, 70:113; Tamm et al, Clin. Cancer Res.2000, 6(5):1796). Overexpression of XIAP in tumor cells has beendemonstrated to confer protection against a variety of pro-apoptoticstimuli and promotes resistance to chemotherapy (LaCasse et al,Oncogene, 1998, 17(25):3247). Consistent with this, a strong correlationbetween XIAP protein levels and survival has been demonstrated forpatients with acute myelogenous leukemia (Tamm et al, supra).Down-regulation of XIAP expression by antisense oligonucleotides hasbeen shown to sensitize tumor cells to death induced by a wide range ofpro-apoptotic agents, both in vitro and in vivo (Sasaki et al, CancerRes., 2000, 60(20):5659; Lin et al, Biochem J., 2001, 353:299; Hu et al,Clin. Cancer Res., 2003, 9(7):2826). Smac/DIABLO-derived peptides havealso been demonstrated to sensitize a number of different tumor celllines to apoptosis induced by a variety of pro-apoptotic drugs (Arnt etal, J. Biol. Chem., 2002, 277(46):44236; Fulda et al, Nature Med., 2002,8(8):808; Guo et al, Blood, 2002, 99(9):3419; Vucic et al, J. Biol.Chem., 2002, 277(14):12275; Yang et al, Cancer Res., 2003, 63(4):831).

Melanoma IAP (ML-IAP) is an IAP not detectable in most normal adulttissues but is strongly upregulated in melanoma (Vucic et al., (2000)Current Bio 10:1359-1366). Determination of protein structuredemonstrated significant homology of the ML-IAP BIR and RING fingerdomains to corresponding domains present in human XIAP, C-IAP1 andC-IAP2. The BIR domain of ML-IAP appears to have the most similaritiesto the BIR2 and BIR3 of XIAP, C-IAP1 and C-IAP2, and appears to beresponsible for the inhibition of apoptosis, as determined by deletionalanalysis. Furthermore, Vucic et al., demonstrated that ML-IAP couldinhibit chemotherapeutic agent induced apoptosis. Agents such asadriamycin and 4-tertiary butylphenol (4-TBP) were tested in a cellculture system of melanomas overexpressing ML-IAP and thechemotherapeutic agents were significantly less effective in killing thecells when compared to a normal melanocyte control. The mechanism bywhich ML-IAP produces an anti-apoptotic activity is in part throughinhibition of caspase 3 and 9. ML-IAP did not effectively inhibitcaspases 1, 2, 6, or 8.

Since apoptosis is a strictly controlled pathway with multipleinteracting factors, the discovery that IAPs themselves are regulatedwas not unusual. In the fruit fly Drosophila, the Reaper (rpr), HeadInvolution Defective (hid) and GRIM proteins physically interact withand inhibit the anti-apoptotic activity of the Drosophila family ofIAPs. In the mammal, the proteins SMAC/DIABLO act to block the IAPs andallow apoptosis to proceed. It was shown that during normal apoptosis,SMAC is processed into an active form and is released from themitochondria into the cytoplasm where it physically binds to IAPs andprevents the IAP from binding to a caspase. This inhibition of the IAPallows the caspase to remain active and thus proceed with apoptosis.Interestingly, sequence homology between the IAP inhibitors shows thatthere is a four amino acid motif in the N-terminus of the processed,active proteins. This tetrapeptide appears to bind into a hydrophobicpocket in the BIR domain and disrupts the BIR domain binding to caspases(Chai et al., (2000) Nature 406:855-862, Liu et al., (2000) Nature408:1004-1008, Wu et al., (2000) Nature 408 1008-1012).

SUMMARY OF THE INVENTION

In one aspect of the present invention there is provided novelinhibitors of IAP proteins having the general formula (I)

wherein

-   X₁, X₂ and X₃ are independently O or S;-   Y is (CHR₇)_(n), O or S; wherein n is 1 or 2 and R₇ is H, halogen,    alkyl, aryl, aralkyl, amino, arylamino, alkylamino, aralkylamino,    alkoxy, aryloxy or aralkyloxy;-   A is a 5-member heterocycle comprising 1 to 4 heteroatoms optionally    substituted with amino, hydroxyl, mercapto, halogen, carboxyl,    amidino, guanidino, alkyl, alkoxy, aryl, aryloxy, acyl, acyloxy,    acylamino, alkoxycarbonylamino, cycloalkyl, alkylthio,    alkylsulfinyl, alkylsulfonyl, aminosulfonyl, alkylaminosulfonyl,    alkylsulfonylamino or a heterocycle; wherein each alkyl, alkoxy,    aryl, aryloxy, acyl, acyloxy, acylamino, cycloalkyl and heterocycle    substitution is optionally substituted with hydroxyl, halogen,    mercapto, carboxyl, alkyl, alkoxy, haloalkyl, amino, nitro, cyano,    cycloalkyl, aryl or a heterocycle;-   R₁ is H or R₁ and R₂ together form a 5-8 member ring;-   R₂ is alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, a    heterocycle or heterocyclylalkyl; each optionally substituted with    hydroxyl, mercapto, halogen, amino, carboxyl, alkyl, haloalkyl,    alkoxy or alkylthio;-   R₃ is H or alkyl;-   R₄ and R₄′ are independently H, hydroxyl, amino, alkyl, aryl,    aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl    wherein each alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,    heteroaryl and heteroarylalkyl is optionally substituted with    halogen, hydroxyl, mercapto, carboxyl, alkyl, alkoxy, amino and    nitro;-   R₅, and R₅′ are each independently H or alkyl;-   R₆, and R₆′ are each independently H, alkyl, aryl or aralkyl;    and salts and solvates thereof.

In another aspect of the invention, there are provided compositionscomprising compounds of formula I and a carrier, diluent or excipient.

In another aspect of the invention, there is provided a method ofinducing apoptosis in a cell comprising introducing into said cell acompound of formula I.

In another aspect of the invention, there is provided a method ofsensitizing a cell to an apoptotic signal comprising introducing intosaid cell a compound of formula I.

In another aspect of the invention, there is provided a method forinhibiting the binding of an IAP protein to a caspase protein comprisingcontacting said IAP protein with a compound of formula I.

In another aspect of the invention, there is provided a method fortreating a disease or condition associated with the overexpression of anIAP protein in a mammal, comprising administering to said mammal aneffective amount of a compound of formula I.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

“Alkyl” means a branched or unbranched, saturated or unsaturated (i.e.alkenyl, alkynyl) aliphatic hydrocarbon group, having up to 12 carbonatoms unless otherwise specified. When used as part of another term, forexample “alkylamino”, the alkyl portion is preferably a saturatedhydrocarbon chain, however also includes unsaturated hydrocarbon carbonchains such as “alkenylamino” and “alkynylamino. Examples of preferredalkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl,iso-butyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl,2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 2,2-dimethylbutyl,n-heptyl, 3-heptyl, 2-methylhexyl, and the like. The terms “lower alkyl”“C₁-C₄ alkyl” and “alkyl of 1 to 4 carbon atoms” are synonymous and usedinterchangeably to mean methyl, ethyl, 1-propyl, isopropyl, cyclopropyl,1-butyl, sec-butyl or t-butyl. Unless specified, substituted, alkylgroups may contain one (preferably), two, three or four substituentswhich may be the same or different. Examples of the above substitutedalkyl groups include, but are not limited to; cyanomethyl, nitromethyl,hydroxymethyl, trityloxymethyl, prop ionyloxymethyl, aminomethyl,carboxymethyl, carboxyethyl, carboxypropyl, alkyloxycarbonylmethyl,allyloxycarbonylaminomethyl, carbamoyloxymethyl, methoxymethyl,ethoxymethyl, t-butoxymethyl, acetoxymethyl, chloromethyl, bromomethyl,iodomethyl, trifluoromethyl, 6-hydroxyhexyl, 2,4-dichloro(n-butyl),2-amino(iso-propyl), 2-carbamoyloxyethyl and the like. The alkyl groupmay also be substituted with a carbocycle group. Examples includecyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, andcyclohexylmethyl groups, as well as the corresponding -ethyl, -propyl,-butyl, -pentyl, -hexyl groups, etc. Preferred substituted alkyls aresubstituted methyls e.g. a methyl group substituted by the samesubstituents as the “substituted C_(n)-C_(m) alkyl” group. Examples ofthe substituted methyl group include groups such as hydroxymethyl,protected hydroxymethyl (e.g. tetrahydropyranyloxymethyl),acetoxymethyl, carbamoyloxymethyl, trifluoromethyl, chloromethyl,carboxymethyl, bromomethyl and iodomethyl.

“Amidine” denotes the group —C(NH)—NHR wherein R is H or alkyl oraralkyl. A preferred amidine is the group —NH—C(NH)—NH₂.

“Amino” denotes primary (i.e. —NH₂), secondary (i.e. —NRH) and tertiary(i.e. —NRR) amines. Preferred secondary and tertiary amines arealkylamine, dialkylamine, arylamine, diarylamine, aralkylamine anddiaralkylamine. Particular preferred secondary and tertiary amines aremethylamine, ethylamine, propylamine, isopropylamine, phenylamine,benzylamine dimethylamine, diethylamine, dipropylamine anddisopropylamine.

“Amino-protecting group” as used herein refers to a derivative of thegroups commonly employed to block or protect an amino group whilereactions are carried out on other functional groups on the compound.Examples of such protecting groups include carbamates, amides, alkyl andaryl groups, imines, as well as many N-heteroatom derivatives which canbe removed to regenerate the desired amine group. Preferred aminoprotecting groups are Boc, Fmoc and Cbz. Further examples of thesegroups are found in T. W. Greene and P. G. M. Wuts, “Protective Groupsin Organic Synthesis”, 2^(nd) ed., John Wiley & Sons, Inc., New York,N.Y., 1991, chapter 7; E. Haslam, “Protective Groups in OrganicChemistry”, J. G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973,Chapter 5, and T. W. Greene, “Protective Groups in Organic Synthesis”,John Wiley and Sons, New York, N.Y., 1981. The term “protected amino”refers to an amino group substituted with one of the aboveamino-protecting groups.

“Aryl” when used alone or as part of another term means a carbocyclicaromatic group whether or not fused having the number of carbon atomsdesignated or if no number is designated, up to 14 carbon atoms.Preferred aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl,naphthacenyl, and the like (see e.g. Lang's Handbook of Chemistry (Dean,J. A., ed) 13^(th) ed. Table 7-2 [1985]) and most preferred phenyl.Substituted phenyl or substituted aryl denotes a phenyl group or arylgroup substituted with one, two, three, four or five, preferably 1-2,1-3 or 1-4 substituents chosen, unless otherwise specified, from halogen(F, Cl, Br, I), hydroxy, protected hydroxy, cyano, nitro, alkyl(preferably C₁-C₆ alkyl), alkoxy (preferably C₁-C₆ alkoxy), benzyloxy,carboxy, protected carboxy, carboxymethyl, protected carboxymethyl,hydroxymethyl, protected hydroxymethyl, aminomethyl, protectedaminomethyl, trifluoromethyl, alkylsulfonylamino, arylsulfonylamino,heterocyclylsulfonylamino, heterocyclyl, aryl, or other groupsspecified. One or more methyne (CH) and/or methylene (CH₂) groups inthese substituents may in turn be substituted with a similar group asthose denoted above. Examples of the term “substituted phenyl” includesbut is not limited to a mono- or di(halo)phenyl group such as2-chlorophenyl, 2-bromophenyl, 4-chlorophenyl, 2,6-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 3-chlorophenyl, 3-bromophenyl,4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl,2-fluorophenyl and the like; a mono- or di(hydroxy)phenyl group such as4-hydroxyphenyl, 3-hydroxyphenyl, 2,4-dihydroxyphenyl, theprotected-hydroxy derivatives thereof and the like; a nitrophenyl groupsuch as 3- or 4-nitrophenyl; a cyanophenyl group, for example,4-cyanophenyl; a mono- or di(lower alkyl)phenyl group such as4-methylphenyl, 2,4-dimethylphenyl, 2-methylphenyl,4-(iso-propyl)phenyl, 4-ethylphenyl, 3-(n-propyl)phenyl and the like; amono or di(alkoxy)phenyl group, for example, 3,4-dimethoxyphenyl,3-methoxy-4-benzyloxyphenyl,3-methoxy-4-(1-chloromethyl)benzyloxy-phenyl, 3-ethoxyphenyl,4-(isopropoxy)phenyl, 4-(t-butoxy)phenyl, 3-ethoxy-4-methoxyphenyl andthe like; 3- or 4-trifluoromethylphenyl; a mono- or dicarboxyphenyl or(protected carboxy)phenyl group such 4-carboxyphenyl, a mono- ordi(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as3-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; amono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl such as2-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or a mono-or di(N-(methylsulfonylamino))phenyl such as3-(N-methylsulfonylamino))phenyl. Also, the term “substituted phenyl”represents disubstituted phenyl groups where the substituents aredifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,2-hydroxy-4-chlorophenyl, and the like, as well as trisubstituted phenylgroups where the substituents are different, for example3-methoxy-4-benzyloxy-6-methyl sulfonylamino,3-methoxy-4-benzyloxy-6-phenyl sulfonylamino, and tetrasubstitutedphenyl groups where the substituents are different such as3-methoxy-4-benzyloxy-5-methyl-6-phenyl sulfonylamino. Preferredsubstituted phenyl groups include the 2-chlorophenyl, 2-aminophenyl,2-bromophenyl, 3-methoxyphenyl, 3-ethoxy-phenyl, 4-benzyloxyphenyl,4-methoxyphenyl, 3-ethoxy-4-benzyloxyphenyl, 3,4-diethoxyphenyl,3-methoxy-4-benzyloxyphenyl,3-methoxy-4-(1-chloromethyl)benzyloxy-phenyl,3-methoxy-4-(1-chloromethyl)benzyloxy-6-methyl sulfonyl aminophenylgroups. Fused aryl rings may also be substituted with any, preferably 1,2 or 3, of the substituents specified herein in the same manner assubstituted alkyl groups.

“Carbocyclyl”, “carbocyclylic”, “carbocycle” and “carbocyclo” alone andwhen used as a moiety in a complex group such as a carbocycloalkylgroup, refers to a mono-, bi-, or tricyclic aliphatic ring having 3 to14 carbon atoms and preferably 3 to 7 carbon atoms which may besaturated or unsaturated, aromatic or non-aromatic. Preferred saturatedcarbocyclic groups include cyclopropyl, cyclobutyl, cyclopentyl andcyclohexyl groups and more preferred are cyclopropyl and cyclohexyl andmost preferred is cyclohexyl. Preferred unsaturated carbocycles arearomatic e.g. aryl groups as previously defined, the most preferredbeing phenyl. The terms “substituted carbocyclyl”, “carbocycle” and“carbocyclo” mean these groups substituted by the same substituents asthe “substituted alkyl” group.

“Carboxy-protecting group” as used herein refers to one of the esterderivatives of the carboxylic acid group commonly employed to block orprotect the carboxylic acid group while reactions are carried out onother functional groups on the compound. Examples of such carboxylicacid protecting groups include 4-nitrobenzyl, 4-methoxybenzyl,3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl,2,4,6-trimethylbenzyl, pentamethylbenzyl, 3,4-methylenedioxybenzyl,benzhydryl, 4,4′-dimethoxybenzhydryl, 2,2′,4,4′-tetramethoxybenzhydryl,alkyl such as t-butyl or t-amyl, trityl, 4-methoxytrityl,4,4′-dimethoxytrityl, 4,4′,4″-trimethoxytrityl, 2-phenylprop-2-yl,trimethylsilyl, t-butyldimethylsilyl, phenacyl, 2,2,2-trichloroethyl,beta-(trimethylsilyl)ethyl, beta-(di(n-butyl)methylsilyl)ethyl,p-toluenesulfonylethyl, 4-nitrobenzylsulfonylethyl, allyl, cinnamyl,1-(trimethylsilylmethyl)prop-1-en-3-yl, and like moieties. The speciesof carboxy-protecting group employed is not critical so long as thederivatized carboxylic acid is stable to the condition of subsequentreaction(s) on other positions of the molecule and can be removed at theappropriate point without disrupting the remainder of the molecule. Inparticular, it is important not to subject a carboxy-protected moleculeto strong nucleophilic bases, such as lithium hydroxide or NaOH, orreductive conditions employing highly activated metal hydrides such asLiAlH₄. (Such harsh removal conditions are also to be avoided whenremoving amino-protecting groups and hydroxy-protecting groups,discussed below.) Preferred carboxylic acid protecting groups are thealkyl (e.g. methyl, ethyl, t-butyl), allyl, benzyl and p-nitrobenzylgroups. Similar carboxy-protecting groups used in the cephalosporin,penicillin and peptide arts can also be used to protect a carboxy groupsubstituents. Further examples of these groups are found in T. W. Greeneand P. G. M. Wuts, “Protective Groups in Organic Synthesis”, 2^(nd) ed.,John Wiley & Sons, Inc., New York, N.Y., 1991, chapter 5; E. Haslam,“Protective Groups in Organic Chemistry”, J. G. W. McOmie, Ed., PlenumPress, New York, N.Y., 1973, Chapter 5, and T. W. Greene, “ProtectiveGroups in Organic Synthesis”, John Wiley and Sons, New York, N.Y., 1981,Chapter 5. The term “protected carboxy” refers to a carboxy groupsubstituted with one of the above carboxy-protecting groups.

“Guanidine” denotes the group —NH—C(NH)—NHR wherein R is H or alkyl oraralkyl. Preferred guanidine is the group —NH—C(NH)—NH₂.

“Hydroxy-protecting group” as used herein refers to a derivative of thehydroxy group commonly employed to block or protect the hydroxy groupwhile reactions are carried out on other functional groups on thecompound. Examples of such protecting groups includetetrahydropyranyloxy, benzoyl, acetoxy, carbamoyloxy, benzyl, andsilylethers (e.g. TBS, TBDPS) groups. Further examples of these groupsare found in T. W. Greene and P. G. M. Wuts, “Protective Groups inOrganic Synthesis”, 2^(nd) ed., John Wiley & Sons, Inc., New York, N.Y.,1991, chapters 2-3; E. Haslam, “Protective Groups in Organic Chemistry”,J. G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, andT. W. Greene, “Protective Groups in Organic Synthesis”, John Wiley andSons, New York, N.Y., 1981. The term “protected hydroxy” refers to ahydroxy group substituted with one of the above hydroxy-protectinggroups.

“Heterocyclic group”, “heterocyclic”, “heterocycle”, “heterocyclyl”, or“heterocyclo” alone and when used as a moiety in a complex group such asa heterocycloalkyl group, are used interchangeably and refer to anymono-, bi-, or tricyclic, saturated or unsaturated, aromatic(heteroaryl) or non-aromatic ring having the number of atoms designated,generally from 5 to about 14 ring atoms, where the ring atoms are carbonand at least one heteroatom (nitrogen, sulfur or oxygen) and preferably1 to 4 heteroatoms. Typically, a 5-membered ring has 0 to 2 double bondsand 6- or 7-membered ring has 0 to 3 double bonds and the nitrogen orsulfur heteroatoms may optionally be oxidized (e.g. SO, SO₂), and anynitrogen heteroatom may optionally be quaternized. Preferrednon-aromatic heterocycles include morpholinyl (morpholino),pyrrolidinyl, oxiranyl, oxetanyl, tetrahydrofuranyl, 2,3-dihydrofuranyl,2H-pyranyl, tetrahydropyranyl, thiiranyl, thietanyl,tetrahydrothietanyl, aziridinyl, azetidinyl, 1-methyl-2-pyrrolyl,piperazinyl and piperidinyl. A “heterocycloalkyl” group is a heterocyclegroup as defined above covalently bonded to an alkyl group as definedabove. Preferred 5-membered heterocycles containing a sulfur or oxygenatom and one to three nitrogen atoms include thiazolyl, in particularthiazol-2-yl and thiazol-2-yl N-oxide, thiadiazolyl, in particular1,3,4-thiadiazol-5-yl and 1,2,4-thiadiazol-5-yl, oxazolyl, preferablyoxazol-2-yl, and oxadiazolyl, such as 1,3,4-oxadiazol-5-yl, and1,2,4-oxadiazol-5-yl. Preferred 5-membered ring heterocycles containing2 to 4 nitrogen atoms include imidazolyl, preferably imidazol-2-yl;triazolyl, preferably 1,3,4-triazol-5-yl; 1,2,3-triazol-5-yl,1,2,4-triazol-5-yl, and tetrazolyl, preferably 1H-tetrazol-5-yl.Preferred benzo-fused 5-membered heterocycles are benzoxazol-2-yl,benzthiazol-2-yl and benzimidazol-2-yl. Preferred 6-memberedheterocycles contain one to three nitrogen atoms and optionally a sulfuror oxygen atom, for example pyridyl, such as pyrid-2-yl, pyrid-3-yl, andpyrid-4-yl; pyrimidyl, preferably pyrimid-2-yl and pyrimid-4-yl;triazinyl, preferably 1,3,4-triazin-2-yl and 1,3,5-triazin-4-yl;pyridazinyl, in particular pyridazin-3-yl, and pyrazinyl. The pyridineN-oxides and pyridazine N-oxides and the pyridyl, pyrimid-2-yl,pyrimid-4-yl, pyridazinyl and the 1,3,4-triazin-2-yl groups, are apreferred group. Substituents for optionally substituted heterocycles,and further examples of the 5- and 6-membered ring systems discussedabove can be found in W. Druckheimer et al., U.S. Pat. No. 4,278,793.

“Heteroaryl” alone and when used as a moiety in a complex group such asa heteroaralkyl group, refers to any mono-, bi-, or tricyclic aromaticring system having the number of atoms designated where at least onering is a 5-, 6- or 7-membered ring containing from one to fourheteroatoms selected from the group nitrogen, oxygen, and sulfur, andpreferably at least one heteroatom is nitrogen (Lang's Handbook ofChemistry, supra). Included in the definition are any bicyclic groupswhere any of the above heteroaryl rings are fused to a benzene ring.Heteroaryls in which nitrogen or oxygen is the heteroatom are preferred.The following ring systems are examples of the heteroaryl (whethersubstituted or unsubstituted) groups denoted by the term “heteroaryl”:thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl,oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl,thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl,thiazinyl, oxazinyl, triazinyl, thiadiazinyl, oxadiazinyl, dithiazinyl,dioxazinyl, oxathiazinyl, tetrazinyl, thiatriazinyl, oxatriazinyl,dithiadiazinyl, imidazolinyl, dihydropyrimidyl, tetrahydropyrimidyl,tetrazolo[1,5-b]pyridazinyl and purinyl, as well as benzo-fusedderivatives, for example benzoxazolyl, benzofuryl, benzothiazolyl,benzothiadiazolyl, benzotriazolyl, benzoimidazolyl and indolyl. Aparticularly preferred group of “heteroaryl” include; 1,3-thiazol-2-yl,4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl,4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl sodium salt,1,2,4-thiadiazol-5-yl, 3-methyl-1,2,4-thiadiazol-5-yl,1,3,4-triazol-5-yl, 2-methyl-1,3,4-triazol-5-yl,2-hydroxy-1,3,4-triazol-5-yl, 2-carboxy-4-methyl-1,3,4-triazol-5-ylsodium salt, 2-carboxy-4-methyl-1,3,4-triazol-5-yl, 1,3-oxazol-2-yl,1,3,4-oxadiazol-5-yl, 2-methyl-1,3,4-oxadiazol-5-yl,2-(hydroxymethyl)-1,3,4-oxadiazol-5-yl, 1,2,4-oxadiazol-5-yl,1,3,4-thiadiazol-5-yl, 2-thiol-1,3,4-thiadiazol-5-yl,2-(methylthio)-1,3,4-thiadiazol-5-yl, 2-amino-1,3,4-thiadiazol-5-yl,1H-tetrazol-5-yl, 1-methyl-1H-tetrazol-5-yl, 1-(1-(dimethylamino)eth-2-yl)-1H-tetrazol-5-yl, 1-(carboxymethyl)-1H-tetrazol-5-yl,1-(carboxymethyl)-1H-tetrazol-5-yl sodium salt, 1-(methylsulfonicacid)-1H-tetrazol-5-yl, 1-(methylsulfonic acid)-1H-tetrazol-5-yl sodiumsalt, 2-methyl-1H-tetrazol-5-yl, 1,2,3-triazol-5-yl,1-methyl-1,2,3-triazol-5-yl, 2-methyl-1,2,3-triazol-5-yl,4-methyl-1,2,3-triazol-5-yl, pyrid-2-yl N-oxide,6-methoxy-2-(n-oxide)-pyridaz-3-yl, 6-hydroxypyridaz-3-yl,1-methylpyrid-2-yl, 1-methylpyrid-4-yl, 2-hydroxypyrimid-4-yl,1,4,5,6-tetrahydro-5,6-dioxo-4-methyl-as-triazin-3-yl,1,4,5,6-tetrahydro-4-(formylmethyl)-5,6-dioxo-as-triazin-3-yl,2,5-dihydro-5-oxo-6-hydroxy-astriazin-3-yl,2,5-dihydro-5-oxo-6-hydroxy-as-triazin-3-yl sodium salt,2,5-dihydro-5-oxo-6-hydroxy-2-methyl-astriazin-3-yl sodium salt,2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl,2,5-dihydro-5-oxo-6-methoxy-2-methyl-as-triazin-3-yl,2,5-dihydro-5-oxo-as-triazin-3-yl,2,5-dihydro-5-oxo-2-methyl-as-triazin-3-yl,2,5-dihydro-5-oxo-2,6-dimethyl-as-triazin-3-yl,tetrazolo[1,5-b]pyridazin-6-yl and8-aminotetrazolo[1,5-b]-pyridazin-6-yl. An alternative group of“heteroaryl” includes; 4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl,4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl sodium salt,1,3,4-triazol-5-yl, 2-methyl-1,3,4-triazol-5-yl, 1H-tetrazol-5-yl,1-methyl-1H-tetrazol-5-yl,1-(1-(dimethylamino)eth-2-yl)-1H-tetrazol-5-yl,1-(carboxymethyl)-1H-tetrazol-5-yl, 1-(carboxymethyl)-1H-tetrazol-5-ylsodium salt, 1-(methylsulfonic acid)-1H-tetrazol-5-yl, 1-(methylsulfonicacid)-1H-tetrazol-5-yl sodium salt, 1,2,3-triazol-5-yl,1,4,5,6-tetrahydro-5,6-dioxo-4-methyl-as-triazin-3-yl,1,4,5,6-tetrahydro-4-(2-formylmethyl)-5,6-dioxo-as-triazin-3-yl,2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl sodium salt,2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl,tetrazolo[1,5-b]pyridazin-6-yl, and8-aminotetrazolo[1,5-b]pyridazin-6-yl.

“Inhibitor” means a compound which reduces or prevents the binding ofIAP proteins to caspase proteins or which reduces or prevents theinhibition of apoptosis by an IAP protein. Alternatively, “inhibitor”means a compound which prevents the binding interaction of X-IAP withcaspases or the binding interaction of ML-IAP with SMAC.

“Pharmaceutically acceptable salts” include both acid and base additionsalts. “Pharmaceutically acceptable acid addition salt” refers to thosesalts which retain the biological effectiveness and properties of thefree bases and which are not biologically or otherwise undesirable,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, carbonic acid, phosphoric acid and the like,and organic acids may be selected from aliphatic, cycloaliphatic,aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes oforganic acids such as formic acid, acetic acid, propionic acid, glycolicacid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid,maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid,citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilicacid, benzoic acid, cinnamic acid, mandelic acid, embonic acid,phenylacetic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicyclic acid and the like.

“Pharmaceutically acceptable base addition salts” include those derivedfrom inorganic bases such as sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum salts andthe like. Particularly preferred are the ammonium, potassium, sodium,calcium and magnesium salts. Salts derived from pharmaceuticallyacceptable organic nontoxic bases includes salts of primary, secondary,and tertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperizine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic non-toxic bases are isopropylamine, diethylamine, ethanolamine,trimethamine, dicyclohexylamine, choline, and caffeine.

The present invention provides novel compounds having the generalformula I:

wherein X, Y, A, R₁, R₂, R₃, R₄, R₄′, R₅, R₅′, R₆ and R₆′ are asdescribed herein.

X₁ and X₂ are each independently O or S. In a preferred embodiment, X₁and X₂ are both O. In another preferred embodiment X₁ and X₂ are both S.In another preferred embodiment, X₁ is S while X₂ is O. In anotherpreferred embodiment, X₁ is O while X₂ is S.

Y is (CHR₇)_(n), O or S; wherein n is 1 or 2 and R₇ is H, halogen,alkyl, aryl, aralkyl, amino, arylamino, alkylamino, aralkylamino,alkoxy, aryloxy or aralkyloxy. In a particular embodiment, Y is CH₂. Ina particular embodiment n is 1. In a particular embodiment n is 1 and Yis CHR₇ wherein R₇ is aralkyloxy, for example benzyloxy. In a particularembodiment n is 1 and Y is CHR₇ wherein R₇ is F. In a particularembodiment n is 1 and Y is CHR₇ wherein R₇ is aralkylamino, for examplebenzylamino. In another particular embodiment Y is O. In anotherparticular embodiment Y is S.

Ring ‘A’ is a 5-member heterocycle comprising 1 to 4 heteroatomsoptionally substituted with amino, hydroxyl, mercapto, halogen,carboxyl, amidino, guanidino, alkyl, alkoxy, aryl, aryloxy, acyl,acyloxy, acylamino, alkoxycarbonylamino, cycloalkyl, alkylthio,alkylsulfinyl, alkylsulfonyl, aminosulfonyl, alkylaminosulfonyl,alkylsulfonylamino or a heterocycle; wherein each alkyl, alkoxy, aryl,aryloxy, acyl, acyloxy, acylamino, cycloalkyl and heterocyclesubstitution is optionally substituted with hydroxyl, halogen, mercapto,carboxyl, alkyl, alkoxy, haloalkyl, amino, nitro, cyano, cycloalkyl,aryl or a heterocycle. In an embodiment, the 5-member heterocycle ring Agroups are optionally substituted with amino, hydroxyl, mercapto,halogen, carboxyl, amidino, guanidino, alkyl, alkoxy, aryl, aryloxy,acyl, acyloxy, acylamino, cycloalkyl or a heterocycle; wherein eachalkyl, alkoxy, aryl, aryloxy, acyl, acyloxy, acylamino, cycloalkyl andheterocycle substitution is optionally substituted with hydroxyl,halogen, mercapto, carboxyl, alkyl, haloalkyl, amino, nitro, cycloalkyl,aryl or a heterocycle. In a particular embodiment ring A is aromatic. Ina particular embodiment ring A has the formula IIa or IIb:

wherein Q₁ is NR₈, O or S; Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, and Q₈, areindependently CR₉ or N; wherein R₉ is H, amino, hydroxyl, mercapto,halogen, carboxyl, amidino, guanidino, alkyl, alkoxy, aryl, aryloxy,acyl, acyloxy, acylamino, cycloalkyl or a heterocycle; wherein eachalkyl, alkoxy, aryl, aryloxy, acyl, acyloxy, acylamino, cycloalkyl andheterocycle substitution is optionally substituted with hydroxyl,halogen, mercapto, carboxyl, alkyl, haloalkyl, amino, nitro, cycloalkyl,aryl or a heterocycle; R₈ is H, alkyl, acyl, aryl, cycloalkyl or aheterocycle; wherein each alkyl, aryl, cycloalkyl and heterocycle isoptionally substituted with hydroxyl, halogen, mercapto, carboxyl,alkyl, haloalkyl, amino, nitro, cycloalkyl, aryl or a heterocycle; andQ₉ is CH or N. In a particular embodiment, ring A is a group of formulaII. In a particular embodiment ring A is a group of formula II whereinQ₄ is CR₉ wherein R₉ is aryl or heteroaryl optionally substituted asdescribed above. In a particular embodiment ring A is a group of formulaII wherein Q₄ is CR₉ and R₉ is phenyl. In a particular embodiment, ringA is a group of formula II wherein Q₄ is CR₉ and R₉ is phenyl and Q₃ isCH or CF. In another embodiment, ring A is a group of formula II whereinQ₄ is CR₉ and R₉ is pyridin-2-yl. In another embodiment, ring A is agroup of formula II wherein Q₄ is CR₉, R₉ is pyridin-2-yl and Q₃ isC-Me.

In another embodiment, ring A according to IIa or IIb is a pyrrole ringoptionally substituted with alkyl, aryl, aralkyl, cycloalkyl,cycloalkylalkyl, a heterocycle or a heterocycle-alkyl optionallysubstituted with halogen hydroxyl, mercapto, carboxyl, alkyl, haloalkyl,amino, nitro, aryl or heteroaryl. In an embodiment ring A is substitutedwith an aryl or heteroaryl group. In a particular embodiment, ring A isselected from the group consisting of:

wherein R₈ is H, alkyl (for example methyl, ethyl or propyl) or acyl(for example acetyl). In a particular embodiment R₈ is H.

In another embodiment ring A is furan optionally substituted with alkyl,aryl, aralkyl, cycloalkyl, cycloalkylalkyl, a heterocycle or aheterocycle-alkyl optionally substituted with halogen hydroxyl,mercapto, carboxyl, alkyl, haloalkyl, amino, nitro, aryl or heteroaryl.In an embodiment ring A is substituted with an aryl or heteroaryl group.In a particular embodiment, ring A is selected from the group consistingof:

In another embodiment ring A is thiophene optionally substituted withalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, a heterocycle or aheterocycle-alkyl optionally substituted with halogen hydroxyl,mercapto, carboxyl, alkyl, haloalkyl, amino, nitro, aryl or heteroaryl.In an embodiment ring A is substituted with an aryl or heteroaryl group.In a particular embodiment, ring A is selected from the group consistingof:

In another embodiment ring A is pyrazole optionally substituted withalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, a heterocycle or aheterocycle-alkyl optionally substituted with halogen hydroxyl,mercapto, carboxyl, alkyl, haloalkyl, amino, nitro, aryl or heteroaryl.In an embodiment ring A is substituted with an aryl or heteroaryl group.In a particular embodiment, ring A is selected from the group consistingof:

wherein R₈ is H, alkyl (for example methyl, ethyl or propyl) or acyl(for example acetyl). In a particular embodiment R₈ is H.

In another embodiment ring A is imidazole optionally substituted withalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, a heterocycle or aheterocycle-alkyl optionally substituted with halogen hydroxyl,mercapto, carboxyl, alkyl, haloalkyl, amino, nitro, aryl or heteroaryl.In an embodiment ring A is substituted with an aryl or heteroaryl group.In a particular embodiment, ring A is selected from the group consistingof:

wherein R₈ is H, alkyl (for example methyl, ethyl or propyl) or acyl(for example acetyl). In a particular embodiment R₈ is H.

In another embodiment ring A is oxazole optionally substituted withalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, a heterocycle or aheterocycle-alkyl optionally substituted with halogen hydroxyl,mercapto, carboxyl, alkyl, haloalkyl, amino, nitro, aryl or heteroaryl.In an embodiment ring A is substituted with an aryl or heteroaryl group.In a particular embodiment, ring A is selected from the group consistingof:

In another embodiment ring A is isoxazole optionally substituted withalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, a heterocycle or aheterocycle-alkyl optionally substituted with halogen hydroxyl,mercapto, carboxyl, alkyl, haloalkyl, amino, nitro, aryl or heteroaryl.In an embodiment ring A is substituted with an aryl or heteroaryl group.In a particular embodiment, ring A is selected from the group consistingof:

In another embodiment ring A is thiazole optionally substituted withalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, a heterocycle or aheterocycle-alkyl optionally substituted with halogen hydroxyl,mercapto, carboxyl, alkyl, haloalkyl, amino, nitro, aryl or heteroaryl.In an embodiment ring A is substituted with an aryl or heteroaryl group.In a particular embodiment, ring A is selected from the group consistingof:

In another embodiment ring A is isothiazole optionally substituted withalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, a heterocycle or aheterocycle-alkyl optionally substituted with halogen hydroxyl,mercapto, carboxyl, alkyl, haloalkyl, amino, nitro, aryl or heteroaryl.In an embodiment ring A is substituted with an aryl or heteroaryl group.In a particular embodiment, ring A is selected from the group consistingof:

In another embodiment ring A is 1,2,3-triazole optionally substitutedwith alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, a heterocycle ora heterocycle-alkyl optionally substituted with halogen hydroxyl,mercapto, carboxyl, alkyl, haloalkyl, amino, nitro, aryl or heteroaryl.In an embodiment ring A is substituted with an aryl or heteroaryl group.In a particular embodiment, ring A is selected from the group consistingof:

wherein R₈ is H, alkyl (for example methyl, ethyl or propyl) or acyl(for example acetyl). In a particular embodiment R₈ is H.

In another embodiment ring A is 1,2,4-triazole optionally substitutedwith alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, a heterocycle ora heterocycle-alkyl optionally substituted with halogen hydroxyl,mercapto, carboxyl, alkyl, haloalkyl, amino, nitro, aryl or heteroaryl.In an embodiment ring A is substituted with an aryl or heteroaryl group.In a particular embodiment, ring A is selected from the group consistingof:

In another embodiment ring A is oxadiazole optionally substituted withalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, a heterocycle or aheterocycle-alkyl optionally substituted with halogen hydroxyl,mercapto, carboxyl, alkyl, haloalkyl, amino, nitro, aryl or heteroaryl.In an embodiment ring A is substituted with an aryl or heteroaryl group.In a particular embodiment, ring A is selected from the group consistingof:

In another embodiment ring A is thiadiazole optionally substituted withalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, a heterocycle or aheterocycle-alkyl optionally substituted with halogen hydroxyl,mercapto, carboxyl, alkyl, haloalkyl, amino, nitro, aryl or heteroaryl.In an embodiment ring A is substituted with an aryl or heteroaryl group.In a particular embodiment, ring A is selected from the group consistingof:

In another embodiment ring A is tetrazole optionally substituted withalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, a heterocycle or aheterocycle-alkyl optionally substituted with halogen hydroxyl,mercapto, carboxyl, alkyl, haloalkyl, amino, nitro, aryl or heteroaryl.In an embodiment ring A is substituted with an aryl or heteroaryl group.In a particular embodiment, ring A is selected from the group consistingof:

In a particular embodiment ring A is:

In a particular embodiment ring A is:

R₁ is H or R₁ and R₂ together form a 5-8 member ring. In a particularembodiment, R₁ is H. In a particular embodiment, R₁ and R₂ together forma 6-member ring. In a particular embodiment, R₁ and R₂ together form a7-member ring. In another particular embodiment, R₁ and R₂ together forman 8-member ring. In another particular embodiment, R₁ and R₂ togetherform a 7-member ring while Y is S. In another particular embodiment, R₁is H, while Y is CH₂. In another particular embodiment, R₁ is H, while Yis S. In another particular embodiment, R₁ is H, while Y is O.

R₂ is alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, a heterocycleor heterocyclylalkyl. In a preferred embodiment R₂ is alkyl orcycloalkyl. In an embodiment, each R₂ group is each optionallysubstituted with hydroxyl, mercapto, halogen, amino, carboxyl, alkyl,haloalkyl, alkoxy or alkylthio; In an embodiment of the invention R₂ ist-butyl, isopropyl, cyclohexyl, cyclopentyl or phenyl. In a particularembodiment, R₂ is cyclohexyl. In another embodiment R₂ istetrahydropyran-4-yl. In another particular embodiment, R₂ is isopropyl(i.e. the valine amino acid side chain). In another particularembodiment, R₂ is t-butyl. In a particular embodiment R₂ is orientedsuch that the amino acid, or amino acid analogue, which it comprises isin the L-configuration.

R₃ is H or alkyl. In a preferred embodiment R₃ is H or methyl, ethyl,propyl or isopropyl. In a particularly preferred embodiment R₃ is H ormethyl. In a most preferred embodiment R₃ is methyl. In anotherparticular embodiment, R₃ is t-butyl. In a preferred embodiment R₃ isoriented such that the amino acid, or amino acid analogue, which itcomprises is in the L-configuration.

R₄ and R₄′ are independently H, hydroxyl, amino, alkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein eachalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl andheteroarylalkyl is optionally substituted with halogen, hydroxyl,mercapto, carboxyl, alkyl, alkoxy, amino and nitro. In a particularembodiment R₄ and R₄′ are both H. In another particular embodiment R₄ ismethyl and R₄′ is H. In a particular embodiment one of R₄ and R₄′ ishydroxyl (OH) while the other is H. In another embodiment, one of R₄ andR₄′ is amino, such as NH₂, NHMe and NHEt, while the other is H. In aparticular embodiment, R₄′ is H and R₄ is H, alkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, heteroaryl or heteroarylalkyl. In aparticular embodiment R₄ is a group selected from the group consistingof:

R₅ and R₅′ are each independently H or alkyl. In a preferred embodiment,R₅ and R₅′ are H or methyl. In a particular embodiment, R₅ is H and R₅′is methyl. In another particular embodiment, R₅ is methyl and R₅′ is H.In another particular embodiment R₅ and R₅′ are both methyl. In anotherparticular embodiment, R₅ and R₅′ are both H.

R₆, and R₆′ are each independently H, alkyl, aryl or aralkyl. In aparticular embodiment, R₆ is alkyl, for example methyl. In anotherparticular embodiment R₆ is aryl, for example phenyl. In anotherparticular embodiment R₆ is aralkyl, for example benzyl. In a particularembodiment R₆ and R₆′ are the same, for example both alkyl, e.g. bothmethyl. In another particular embodiment R₆ is methyl and R₆′ is H.

Compounds of the invention contain one or more asymmetric carbon atoms.Accordingly, the compounds may exist as diastereomers, enantiomers ormixtures thereof. The syntheses of the compounds may employ racemates,diastereomers or enantiomers as starting materials or as intermediates.Diastereomeric compounds may be separated by chromatographic orcrystallization methods. Similarly, enantiomeric mixtures may beseparated using the same techniques or others known in the art. Each ofthe asymmetric carbon atoms may be in the R or S configuration and bothof these configurations are within the scope of the invention.Preferably, compounds of the invention have the following stereochemicalconfiguration of formula I′

wherein X, Y, A, R₁, R₂, R₃, R₄, R₄′, R₅, R₅′, R₆ and R₆′ are asdescribed herein.

The invention also encompasses prodrugs of the compounds describedabove. Suitable prodrugs where applicable include known amino-protectingand carboxy-protecting groups which are released, for examplehydrolyzed, to yield the parent compound under physiologic conditions. Apreferred class of prodrugs are compounds in which a nitrogen atom in anamino, amidino, aminoalkyleneamino, iminoalkyleneamino or guanidinogroup is substituted with a hydroxy (OH) group, an alkylcarbonyl (—CO—R)group, an alkoxycarbonyl (—CO—OR), an acyloxyalkyl-alkoxycarbonyl(—CO—O—R—O—CO—R) group where R is a monovalent or divalent group and asdefined above or a group having the formula —C(O)—O—CP1P2-haloalkyl,where P1 and P2 are the same or different and are H, lower alkyl, loweralkoxy, cyano, halo lower alkyl or aryl. Preferably the nitrogen atom isone of the nitrogen atoms of the amidino group of the compounds of theinvention. These prodrug compounds are prepared reacting the compoundsof the invention described above with an activated acyl compound to bonda nitrogen atom in the compound of the invention to the carbonyl of theactivated acyl compound. Suitable activated carbonyl compounds contain agood leaving group bonded to the carbonyl carbon and include acylhalides, acyl amines, acyl pyridinium salts, acyl alkoxides, inparticular acyl phenoxides such as p-nitrophenoxy acyl, dinitrophenoxyacyl, fluorophenoxy acyl, and difluorophenoxy acyl. The reactions aregenerally exothermic and are carried out in inert solvents at reducedtemperatures such as −78 to about 50 C. The reactions are usually alsocarried out in the presence of an inorganic base such as potassiumcarbonate or sodium bicarbonate, or an organic base such as an amine,including pyridine, triethylamine, etc. One manner of preparing prodrugsis described in U.S. Ser. No. 08/843,369 filed Apr. 15, 1997(corresponding to PCT publication WO9846576) the contents of which areincorporated herein by reference in their entirety.

Particular compounds of formula I include the following:

Synthesis

Compounds of the invention are prepared using standard organic synthetictechniques from commercially available starting materials and reagents.It will be appreciated that synthetic procedures employed in thepreparation of compounds of the invention will depend on the particularsubstituents present in a compound and that various protection anddeprotection may be required as is standard in organic synthesis. In ageneral synthetic scheme compounds of the invention may be preparedusing typical peptide chemistry techniques by coupling the amino acidresidue analogues with typical amide coupling procedures. In scheme 1,amine-protected amino acid residue analogues are coupled and deprotectedsequentially to give the final compounds.

It will be appreciated that the amino acid analogs may be coupled anyorder and may be prepared using solid phase support which is routine inthe art.

Amine substituted ring A which serves as an intermediate for preparingcompounds of the invention are commercially available or else areprepared from commercially available reagents employing standard organicchemistry techniques. For example, 1-Aryl-5-aminotetrazoles, such asphenyl-5-aminotetrazole, may be prepared according to scheme 2 fromcommercially available phenyl thiourea by reacting with sodium azide andmercuric chloride.

3-Aryl-5-amino-1,2,3-triazoles, such as3-Phenyl-3H-[1,2,3]triazol-4-ylamine, may be prepared according to theprocedures described in J. Org. Chem., 1981, 46:856-9 and illustrated inscheme 3 below by reacting phenylamine with aminoacetonitrile.

Similarly, 5-Amino-1-phenyl-1H-[1,2,3]triazole-4-carbonitrile may beprepared by reacting phenylamine with 2-amino-malononitrile asillustrated in scheme 4.

4-Aryl-5-amino-1,2,5-oxadiazoles, such as 4-phenyl-furazan-3-ylamine,may be prepared according to the procedures described in Lakhan et al,(Indian Journal of Chemistry, Section B: Organic Chemistry IncludingMedicinal Chemistry (1987), 26B(7), 690-2) and illustrated in Scheme 5by reacting benzoyl cyanide with hydroxylamine.

4-Aryl-3-amino-1,2,4-triazoles, such as4-phenyl-4H-[1,2,4]triazol-3-ylamine, may be prepared by reactingphenylisothiocyanate with hydrazinecarboximidamide to give5-amino-4-phenyl-4H-[1,2,4]triazole-3-thiol in which the thiol group maybe removed with Raney nickel catalyst as illustrated in scheme 6.

4-Aryl-5-amino-1,2,3-triazoles such as3,5-diphenyl-3H-[1,2,3]triazol-4-ylamine according to the proceduresdescribed in J. Org. Chem., 1990, 55:3351-62 and illustrated in scheme7, by reacting benzeneacetonitrile with azidobenzene (or alternativelytrimethylsilylazide, TMS-N₃).

4-Aryl-3-aminopyrazoles such as 4-phenyl-2H-pyrazol-3-ylamine may beprepared according to the procedures described in patent EP269,859 andillustrated in scheme 8, by reacting benzeneacetonitrile withorthoformic acid triethyl ester to give 3-oxo-2-phenyl-propionitrilewhich is reacted with hydrazine.

Various hydrazines and derivatives of benzeneacetonitrile can be used toprepare substituted-4-aryl-3-aminopyrazoles as illustrated in scheme 9.

1-Aryl-5-aminopyrazoles such as 2-phenyl-2H-pyrazol-3-ylamine may beprepared by reacting phenylhydrazine with 3-oxo-propionitrile. Variousnitriles can be used to introduce substitution at the 3-position of thepyrazole ring as illustrated in scheme 10.

3-Aryl-4-aminoimidazoles such as 3-phenyl-3H-imidazol-4-ylamine may beprepared by reacting phenylamine with aminoacetonitrile and orthoformicacid triethyl ester as illustrated in scheme 11. Substitution at the2-position of the imidazole can be introduced using analogs of theorthoformic acid triethylester as follows.

5-Aryl-4-aminoimidazoles such as 5-phenyl-3H-imidazol-4-ylamine may beprepared by reacting formamidine with aminophenylacetonitrile asillustrated in scheme 12. Substitution at the 2-position of theimidazole ring can be introduced using analogs of the formamidine.

4-Aryl-[1,2,3]thiadiazol-5-ylamines such as4-phenyl-[1,2,3]thiadiazol-5-ylamine may be prepared by procedureillustrated in scheme 13. 2-bromo-1-phenyl-ethanone is reacted withlithium phthalimide and the substitution product is reacted withhydrazinecarboxylate ethyl ester. The resulting hydrazinecarboxylateethyl ester is cyclized to form a thiadiazole by reacting with thionylchloride followed by removal of the phthalimide group with hydrazine.

Compounds of the invention in which R₄ or R₄′ are other than H may beprepared according to standard organic chemistry techniques, for exampleby reductive amination in which a starting amino acid residue analoge.g. NH₂—CH(R₃)—C(O)—OH is reacted with a suitable aldehyde or ketone togive the desired R₄ and R₄′ substituents. See scheme 14. The resultingR₄/R₄′ substituted amino acid intermediate can then be conjugated to thenext amino acid intermediate or the remainder of the compound usingstandard peptide coupling procedures.

In a particular embodiment, alanine is reacted with1-methylindole-2-carboxaldehyde and reduced with sodium cyanoborohydridedissolved in 1% HOAc/DMF to give the N-substituted alanine residue whichmay be used in preparing compounds of the invention. See scheme 15.

Alternatively, the reductive amination procedure to introduce R₄/R₄′substituents is the final step in the preparation of the compound.

When compounds of the invention incorporate R₄ or R₄′ substituents otherthan H, they may also be prepared by substitution of a suitable acidintermediate which incorporates a leaving group with a desired amine.For example Br—CH(R₃)—C(O)—OH is substituted with an amine R₄—NH₂ orR₄—NH—R₄′ according to scheme 16.

Alternatively, the substitution reaction introducing R₄ or R₄′substituents may be performed as a final step in the preparation of thecompound as illustrated in scheme 17.

In a particular embodiment, 2-bromopropionic acid is reacted with thefollowing amines dissolved in DMF and bubbled for until substitution iscomplete to form N-substituted alanine residues:

Compounds of the invention in which any one or more of X₁, X₂ and X₃ aresulfur, i.e. the compound incorporates a thioamide, may be preparedaccording to established organic chemistry techniques. For example,compounds in which X₂ is sulfur can be prepared according to scheme 18starting from an Fmoc protected amino acid residue analogNH₂—CH(R₂)—COOH which is dissolved in THF and cooled to −25° C., withaddition of DIPEA followed by addition of isobutylchloroformate. After10 minutes, the diamine, 4-nitrobenzene-1,2-diamine, is added and thereaction mixture is continuously stirred at −25° C. for 2 hours, then atroom temperature overnight. THF is vacuumed off and the mixture is thensubjected to flash chromatography using 50% EtOAc/Hexane to yield theproduct. The Fmoc-alanine derivative, phosphorus pentasulfide and sodiumcarbonate are mixed in THF and stirred overnight. The solution isconcentrated and direct chromatography using 80% EtOAc/Hexane yields theactivated thioalanine. The activated thioalanine and sodium nitrite arethen mixed in acetic acid and diluted with H₂O. The resultingprecipitant is filtered and dried to yield the product. The thioalanineis coupled to an OH-protected proline amino acid residue analog bydissolving both in DMF. The thioamide product may then be deprotectedwith 20% PIP/DMA for 15 minutes and used to conjugate to theR₄/R₄′-N—CH(R₃)—COOH amino acid residue analog followed byOH-deprotection and coupling to an amino-substituted A ringintermediate. Alternatively the Fmoc-protected thioamide is firstcoupled to an amino substituted A ring intermediate followed by Fmocdeprotection and subsequent coupling to the R₄/R₄′—N—CH(R₃)—COOH aminoacid residue analog.

Utility

The compounds of the invention inhibit the binding of IAP proteins tocaspases, in particular X-IAP binding interaction with caspases 3 and 7.The compounds also inhibit the binding of ML-IAP to Smac protein.Accordingly, the compounds of the invention are useful for inducingapoptosis in cells or sensitizing cells to apoptotic signals, inparticular cancer cells. Compounds of the invention are useful forinducing apoptosis in cells that overexpress IAP proteins.Alternatively, compounds of the invention are useful for inducingapoptosis in cells in which the mitochondrial apoptotic pathway isdisrupted such that release of Smac from ML-IAP proteins is inhibited,for example by up regulation of Bcl-2 or down regulation of Bax/Bak.More broadly, the compounds can be used for the treatment of all cancertypes which fail to undergo apoptosis. Examples of such cancer typesinclude neuroblastoma, intestine carcinoma such as rectum carcinoma,colon carcinoma, familiary adenomatous polyposis carcinoma andhereditary non-polyposis colorectal cancer, esophageal carcinoma, labialcarcinoma, larynx carcinoma, hypopharynx carcinoma, tong carcinoma,salivary gland carcinoma, gastric carcinoma, adenocarcinoma, medullarythyroidea carcinoma, papillary thyroidea carcinoma, renal carcinoma,kidney parenchym carcinoma, ovarian carcinoma, cervix carcinoma, uterinecorpus carcinoma, endometrium carcinoma, chorion carcinoma, pancreaticcarcinoma, prostate carcinoma, testis carcinoma, breast carcinoma,urinary carcinoma, melanoma, brain tumors such as glioblastoma,astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermaltumors, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, acutelymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), acutemyeloid leukemia (AML), chronic myeloid leukemia (CML), adult T-cellleukemia lymphoma, hepatocellular carcinoma, gall bladder carcinoma,bronchial carcinoma, small cell lung carcinoma, non-small cell lungcarcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma,choroidea melanoma, seminoma, rhabdomyo sarcoma, craniopharyngeoma,osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma,Ewing sarcoma and plasmocytoma.

Compounds of the invention are useful for sensitizing cells to apoptoticsignals. Accordingly, the compounds may be administered prior to,concomitantly with, or following administration of radiation therapy orcytostatic or antineoplastic chemotherapy. Suitable cytostaticchemotherapy compounds include, but are not limited to (i)antimetabolites, such as cytarabine, fludarabine,5-fluoro-2′-deoxyuiridine, gemcitabine, hydroxyurea or methotrexate;(ii) DNA-fragmenting agents, such as bleomycin, (iii) DNA-crosslinkingagents, such as chlorambucil, cisplatin, cyclophosphamide or nitrogenmustard; (iv) intercalating agents such as adriamycin (doxorubicin) ormitoxantrone; (v) protein synthesis inhibitors, such as L-asparaginase,cycloheximide, puromycin or diphtheria toxin; (Vi) topoisomerase Ipoisons, such as camptothecin or topotecan; (vii) topoisomerase IIpoisons, such as etoposide (VP-16) or teniposide; (viii)microtubule-directed agents, such as colcemid, colchicine, paclitaxel,vinblastine or vincristine; (ix) kinase inhibitors such as flavopiridol,staurosporin, STI571 (CPG 57148B) or UCN-01 (7-hydroxystaurosporine);(x) miscellaneous investigational agents such as thioplatin, PS-341,phenylbutyrate, ET-18-OCH₃, or farnesyl transferase inhibitors(L-739749, L-744832); polyphenols such as quercetin, resveratrol,piceatannol, epigallocatechine gallate, theaflavins, flavanols,procyanidins, betulinic acid and derivatives thereof; (xi) hormones suchas glucocorticoids or fenretinide; (xii) hormone antagonists, such astamoxifen, finasteride or LHRH antagonists. In a preferred embodiment,compounds of the present invention are coadministered with a cytostaticcompound selected from the group consisting of cisplatin, doxorubicin,taxol, taxotere and mitomycin C. Most preferred, the cytostatic compoundis doxorubicin.

Another class of active compounds which can be used in the presentinvention are those which are able to sensitize for or induce apoptosisby binding to death receptors (“death receptor agonists”). Such agonistsof death receptors include death receptor ligands such as tumor necrosisfactor a (TNF-α), tumor necrosis factor β (TNF-β, lymphotoxin-α),LT-β(lymphotoxin-β), TRAIL (Apo2L, DR4 ligand), CD95 (Fas, APO-1)ligand, TRAMP (DR3, Apo-3) ligand, DR6 ligand as well as fragments andderivatives of any of said ligands. Preferably, the death receptorligand is TNF-α. More preferably the death receptor ligand isApo2L/TRAIL. Furthermore, death receptors agonists comprise agonisticantibodies to death receptors such as anti-CD95 antibody, anti-TRAIL-R1(DR4) antibody, anti-TRAIL-R2 (DR5) antibody, anti-TRAIL-R3 antibody,anti-TRAIL-R4 antibody, anti-DR6 antibody, anti-TNF-R1 antibody andanti-TRAMP (DR3) antibody as well as fragments and derivatives of any ofsaid antibodies.

For the purpose of sensitizing cells for apoptosis, the compounds of thepresent invention can be also used in combination with radiationtherapy. The phrase “radiation therapy” refers to the use ofelectromagnetic or particulate radiation in the treatment of neoplasia.Radiation therapy is based on the principle that high-dose radiationdelivered to a target area will result in the death of reproducing cellsin both tumor and normal tissues. The radiation dosage regimen isgenerally defined in terms of radiation absorbed dose (rad), time andfractionation, and must be carefully defined by the oncologist. Theamount of radiation a patient receives will depend on variousconsideration but the two most important considerations are the locationof the tumor in relation to other critical structures or organs of thebody, and the extent to which the tumor has spread. Examples ofradiotherapeutic agents are provided in, but not limited to, radiationtherapy and is known in the art (Hellman, Principles of RadiationTherapy, Cancer, in Principles I and Practice of Oncology, 24875 (Devitaet al., 4th ed., vol 1, 1993). Recent advances in radiation therapyinclude three-dimensional conformal external beam radiation, intensitymodulated radiation therapy (IMRT), stereotactic radiosurgery andbrachytherapy (interstitial radiation therapy), the latter placing thesource of radiation directly into the tumor as implanted “seeds”. Thesenewer treatment modalities deliver greater doses of radiation to thetumor, which accounts for their increased effectiveness when compared tostandard external beam radiation therapy.

Ionizing radiation with beta-emitting radionuclides is considered themost useful for radiotherapeutic applications because of the moderatelinear energy transfer (LET) of the ionizing particle (electron) and itsintermediate range (typically several millimeters in tissue). Gamma raysdeliver dosage at lower levels over much greater distances. Alphaparticles represent the other extreme, they deliver very high LETdosage, but have an extremely limited range and must, therefore, be inintimate contact with the cells of the tissue to be treated. Inaddition, alpha emitters are generally heavy metals, which limits thepossible chemistry and presents undue hazards from leakage ofradionuclide from the area to be treated. Depending on the tumor to betreated all kinds of emitters are conceivable within the scope of thepresent invention.

Furthermore, the present invention encompasses types of non-ionizingradiation like e.g. ultraviolet (UV) radiation, high energy visiblelight, microwave radiation (hyperthermia therapy), infrared (IR)radiation and lasers. In a particular embodiment of the presentinvention UV radiation is applied.

The invention also includes pharmaceutical compositions or medicamentscontaining the compounds of the invention and a therapeutically inertcarrier, diluent or excipient, as well as methods of using the compoundsof the invention to prepare such compositions and medicaments.Typically, the compounds of formula I used in the methods of theinvention are formulated by mixing at ambient temperature at theappropriate pH, and at the desired degree of purity, withphysiologically acceptable carriers, i.e., carriers that are non-toxicto recipients at the dosages and concentrations employed into agalenical administration form. The pH of the formulation depends mainlyon the particular use and the concentration of compound, but preferablyranges anywhere from about 3 to about 8. Formulation in an acetatebuffer at pH 5 is a suitable embodiment.

The inhibitory compound for use herein is preferably sterile. Thecompound ordinarily will be stored as a solid composition, althoughlyophilized formulations or aqueous solutions are acceptable.

The composition of the invention will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“effective amount” of the compound to be administered will be governedby such considerations, and is the minimum amount necessary to inhibitIAP interaction with caspases, induce apoptosis or sensitize a malignantcell to an apoptotic signal. Such amount is preferably below the amountthat is toxic to normal cells, or the mammal as a whole.

Generally, the initial pharmaceutically effective amount of the compoundof the invention administered parenterally per dose will be in the rangeof about 0.01-100 mg/kg, preferably about 0.1 to 20 mg/kg of patientbody weight per day, with the typical initial range of compound usedbeing 0.3 to 15 mg/kg/day. Oral unit dosage forms, such as tablets andcapsules, preferably contain from about 25 to about 1000 mg of thecompound of the invention.

The compound of the invention may be administered by any suitable means,including oral, topical, transdermal, parenteral, subcutaneous,intraperitoneal, intrapulmonary, and intranasal, and, if desired forlocal treatment, intralesional administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. An example of a suitable oral dosage formis a tablet containing about 25 mg, 50 mg, 100 mg, 250 mg, or 500 mg ofthe compound of the invention compounded with about 90-30 mg anhydrouslactose, about 5-40 mg sodium croscarmellose, about 5-30 mgpolyvinylpyrrolidone (PVP) K30, and about 1-10 mg magnesium stearate.The powdered ingredients are first mixed together and then mixed with asolution of the PVP. The resulting composition can be dried, granulated,mixed with the magnesium stearate and compressed to tablet form usingconventional equipment. An aerosol formulation can be prepared bydissolving the compound, for example 5-400 mg, of the invention in asuitable buffer solution, e.g. a phosphate buffer, adding a tonicifier,e.g. a salt such sodium chloride, if desired. The solution is typicallyfiltered, e.g. using a 0.2 micron filter, to remove impurities andcontaminants.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. Abbreviations used herein are as follows:

ACN: acetonitrile;

Chg: cyclohexylglycine;

DCM: dichloromethane

DIPEA: diisopropylethylamine;

DMAP: 4-dimethylaminopyridine;

DME: 1,2-dimethoxyethane;

DMF: dimethylformamide;

DMSO: dimethylsulfoxide

EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;

EEDQ: 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline

LCMS: liquid chromatography mass spectrometry;

HATU: O-(7-Azobenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate;

HOBt: N-Hydroxybenzotriazole

HBTU: 2-(1H-Benzotriazol-1-yl)-1,1,3,3-Tetramethyl-uroniumHexafluorophosphate

HPLC: high performance liquid chromatography;

NBS: N-bromosuccinamide;

TASF: tris(dimethylamino)sulfonium difluorotrimethylsilicate;

TEA: triethylamine;

TFA: trifluoroacetate;

THF: tetrahydrofuran;

Example 16-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-5-oxo-octahydro-thiazolo[3,2-a]azepine-3-carboxylicacid ethyl ester

To a stirred solution of N-(Diphenylmethylene) glycine t-butyl ester 1(3.0 g, 10.1 mmol) and chiral catalystO-Allyl-N-(9-anthracenylmethyl)-cinchonidium bromide (613 mg, 1.0 mmol)in dry DCM (30 mL) was added cesium hydroxide (17 g, 101 mmol). Thereaction was cooled to −78° C. in a dry ice acetone bath and4-bromo-1-butene was added dropwise. After addition the reaction wasstirred vigorously under N₂ at −48° C. for 48 hours. Ethyl ether wasadded followed by H₂O. The organic layer was separated and washed 2×with H₂O, 1× brine, dried with MgSO₄ and concentrated. The product waspurified by SiO₂ chromatography over a gradient of 0-10% EtOAc inHexanes to give 2 in 65% yield.

To a stirred solution of 2 (1.52 g, 4.3 mmol) in dry MeOH (50 mL) wasadded NaOAc (720 mg, 8.6 mmol) and NH₂OH.HCl (540 mg, 7.6 mmol). Stirredunder N₂ at room temperature for 2 hours. DCM and 0.1 N NaOH were added.The aqueous layer was separated and extracted 3× with DCM, dried withNa₂SO₄ and the DCM fractions were combined and concentrated. The productwas purified by SiO₂ chromatography, 0-10% MeOH in DCM with 0.05% TEA togive 3 in 70% yield.

To a solution of 3 (610 mg, 3.3 mmol) in dry DCM (20 mL) was addedtriethylamine (550 μL, 3.9 mmol) and benzyl chloroformate (550 μL, 3.9mmol). The reaction was stirred at room temperature for 2 hours. Thesolution was concentrated and purified by SiO₂ chromatography over agradient of 0-30% EtOAc in Hexanes to give 4 in 66% yield.

To a stirred solution of 4 (577 mg, 1.8 mmol) in THF (20 mL) under N₂was added BH₃.THF. After 1 hour 3 N NaOH (300 μL, 0.9 mmol) and H₂O₂(306 μL, 2.7 mmol) was added. The reaction was stirred overnight andsubsequently diluted with H₂O, extracted 2× with ethyl ether, dried withMgSO₄ and concentrated. The product was purified by SiO₂ chromatographyover a gradient of 10-45% EtOAc in Hexanes to give 5 in 50% yield.

To a stirred solution of 5 (71 mg, 0.21 mmol) in MeOH (2 mL) under 1 atmH₂ 10% palladium hydroxide on carbon (30 mg) was added. The reaction wascomplete after 30 minutes. The reaction was filtered over Celite andconcentrated to give 6 in quantitative yield.

To 6 (42 mg, 0.21 mmol) in ACN (2 mL) carbethoxyphthalimide (50 mg, 0.23mmol) was added with DIPEA (40 μL, 0.23 mmol) and stirred at roomtemperature for 2 hours. H₂O (1 mL) was added and stirred for anadditional 10 minutes. The ACN was evaporated off and DCM and 10% citricacid were added. The aqueous layer was separated and extracted 3× withDCM, the DCM portions were combined, dried with Na₂SO₄, and concentratedto give 7 in 95% yield.

Oxalyl chloride (561 μL, 6.60 mmol) was dissolved in DCM (35 mL), cooledto −78° C., stirred for 5 minutes followed by addition of a solution ofdimethylsulfoxide (870 μL, 12.3 mmol) in DCM (2.5 mL). After stirringfor 5 minutes 7 (1.05 g, 3.15 mmol) in dichloromethane (20 mL) was addedfollowed by triethylamine (2.37 mL, 17.0 mmol). The reaction was slowlywarmed to room temperature. DCM and H₂O were added, the aqueous layerseparated and extracted 2× with DCM. The DCM portions were combined,filtered through Na₂SO₄, and concentrated to give 8 in 95% yield.

L-cysteine ethyl ester hydrochloride (643 mg, 3.5 mmol) and potassiumacetate (343 mg, 3.5 mmol) were dissolved in stirring EtOH (13 mL), andcooled to 0° C. in an ice water bath. Compound 8 was dissolved in EtOH(13 mL) and added. The reaction was stirred at 0° C. for 4 hours, LCMSconfirmed the conversion of 8 into two diastereomeric products. Thereaction was filtered, EtOH evaporated, redissolved in DCM and washedwith brine, dried with MgSO₄ and concentrated to give a 1:1 mixture ofdiastereomers 9 in quantitative yield.

The diastereomers were redissolved in 1:1 TFA:DCM (10 mL) and stirredfor 1 hour at room temperature. LCMS showed complete conversion to 10.The reaction was concentrated to give 10 in 95% yield for the twodiastereomers.

To a stirred solution of 10 (675 mg, 1.67 mmol) in THF (20 mL), EEDQ(619 mg, 2.50 mmol) was added. Stirred at room temperature for 2 days.The THF was removed under reduced pressure, the product redissoved inEtOAc. The organic layer was washed with 0.5 N HCl, 0.5% NaHCO₃, H₂O,brine. The EtOAc solution was dried with MgSO₄ and concentrated. Theproduct was purified via reverse phase HPLC 10-70% ACN in H₂O to givetwo diastereomers 11, 20% yield for diastereomer 1 and 18% yield fordiastereomer 2.

Example 21-[2-Cyclohexyl-2-(2-methylamino-propionylamino)-acetyl]-pyrrolidine-2-carboxylicacid (2-phenyl-2H-pyrazol-3-yl)-amide

A solution of Boc-MeAla-Chg-Pro-OH (47.0 mg, 0.107 mmol) and pyridine(26 μl, 0.32 mmol) in anhydrous dichloromethane (300 μl) was cooled to0° C. and a solution of oxalyl chloride in dichloromethane (54 μl, 2.0M, 0.11 mmol) was added dropwise over 10 minutes. The mixture wasstirred at 0° C. for 15 minutes, then at ambient temperature for 45minutes, and a solution of 5-amino-1-phenylpyrazole (15.9 mg, 0.100mmol; TCI America catalog #A0174) and pyridine (15.5 μl, 0.191 mmol) indichloromethane (0.5 ml) was added. The resulting mixture was stirred atambient temperature for 16 hours, diluted with dichloromethane to 20 ml,and washed with 0.2 N aqueous sodium hydroxide (20 ml). The organicphase was dried (MgSO₄) and concentrated under reduced pressure. Thecrude product was purified by column chromatography (silica gel, 60%ethyl acetate in hexanes, then 100% ethyl acetate) to yield a yellowoil: m/z 581 (MAO The oil was treated with 5% trifluoroacetic acid indichloromethane (2 ml), and after 18 hours the solvent was removed invacuo. The resulting oil (29.3 mg, 57% yield over 2 steps) was furtherpurified by reversed-phase HPLC to yield the product (TFA salt, 9.6 mg,15% yield): m/z 481 (M+H⁺), 503 (M+Na⁺).

Example 3 4-Phenyl-[1,2,3]thiadiazol-5-ylamine

2-bromoacetophenone was dissolved in DMF (3 vol) and potassiumphthalimide (1.1 eq.) was added. The reaction, initially mildlyexothermic, was stirred overnight at room temperature.

The DMF was removed in vacuo and the reaction was diluted with DCM (˜3vol) followed by 0.1N NaOH (˜3 vol; 1:1 aq/org) and stirred vigorouslythen extracted. The organic layer, containing some solid material, wasconcentrated in vacuo and the resulting solid was slurried in diethylether and collected by suction filtration to give (a) as a whitecrystalline solid in −95% yield.

Compound (a), ethyl carbazate (1.5 eq) and TsOH—H,O (0.1 eq) werecombined in toluene (5 vol) and heated to reflux using a Dean-Stark trapto remove water. The solution turned a dark red color and was completeby TLC in −2 hrs. Approximately half of the toluene was removed bydistillation, the solution was cooled to r.t. and concentrated in vacuo.The resulting solid was slurried in EtOH (the minimum volume necessaryfor stirring), heated to reflux for 30 min and then cooled on ice tofacilitate precipitation of both isomers. The solid was collected bysuction filtration, washed with cold EtOH and dried under vacuum to giveboth isomers of compound (b) as an off-white solid in ˜90% yield.

To ice-cooled thionyl chloride (4 eq, ˜0.85 vol) was added, portion-wise(in order to control the exotherm), the isomer mixture of (b). The icebath was removed and the reaction warmed to r.t and stirred overnight.Thionyl chloride was removed in vacuo, DCM (1 vol) was added and thereaction was stirred with 0.1M NaOH (1 vol; 1:1 aq/org). The suspensionwas extracted and the organics were concentrated in vacuo, slurried inrefluxing EtOAc (the minimum volume necessary for easy stirring) for 30min, cooled to r.t., collected by suction filtration and washed with aminimum of cold EtOAc to give (c) as an off-white crystalline solid in˜80% yield.

A solution of hydrazine hydrate (2.4 eq) in EtOH (1 vol) was addeddropwise to a refluxing solution of (c) in EtOH (8 vol). A precipitateformed almost immediately and the reaction was completed by TLC in ˜3hrs. The solution was cooled to r.t. and the phthalimide cleavageby-product was filtered away and washed with DCM. The EtOH/DCM filtratewas concentrated in vacuo until crystal formation was observed. Thissuspension was stirred overnight and the crystalline/solid mixture wascollected by suction filtration and washed with cold EtOH until coloredimpurities were removed, giving the thiadiazole amine (d) in ˜75% yieldas an off-white crystalline solid.

Example 41-[2-Cyclohexyl-2-(2-methylamino-propionylamino)-acetyl]-pyrrolidine-2-carboxylicacid (4-phenyl-[1,2,3]thiadiazol-5-yl)-amide

Boc-L-Pro (2 eq), HOBt (1.9 eq), EDC-HCl (1.9 eq) and DIPEA (5 eq) weredissolved in DMF (10-15 vol). To this was then added the thiadiazoleamine (d). The reaction, initially mildly exothermic, was heated to 75°C. and stirred overnight, cooled to room temperature and the DMF waspartially removed in vacuo. Dilution with EtOAc (10-15 vol) was followedby washing with 1M HCl (2×), NaHCO₃ (1×), and brine (1×) (1:1 aq/org).The organic layer was concentrated in vacuo and the resulting solid wasslurried in refluxing MeCN (a minimum volume necessary for easystirring) for 30 min and then cooled to r.t. Suction filtration gaveBoc-protected conjugation product as an off-white crystalline solid in˜77% yield. The Boc-protected product was suspended in a solution of 4MHCl/dioxane (4-5 eq acid) and MeCN (1 vol eq to the dioxane solution)and stirred at r.t. until LCMS indicated complete deprotection, ˜1 hr.The reaction mixture was concentrated in vacuo and the resulting solidwas vigorously slurried in refluxing MeCN (a minimum volume necessaryfor easy stirring), cooled to r.t., and the solid collected by suctionfiltration and washed with cold MeCN until residual color was removedfrom the cake to yield the HCl salt (e) as an off-white solid inapproximately quantitative yield.

The HCl salt (e) was dissolved in DMF (10-15 vol) and DIPEA (5 eq). Tothis was added the Boc-L-Chg (1.5 eq), HOBt (1.4 eq) and EDC-HCl (1.4eq). Coupling was complete after ˜2 hrs by LCMS. The reaction wasdiluted with EtOAc (15 vol) and washed with 1M HCl (2×), NaHCO₃ (1×),and brine (1×) (1:1 aq/org). The organic extract was dried over sodiumsulfate and concentrated in vacuo. The resulting solid is slurried inEtOH/Hexane (20:80) (a minimum volume necessary for easy stirring) andfiltered to give Boc-protected conjugate product as a fluffy white solidin ˜80% yield. The Boc-protected was dissolved in a solution of 4MHCl/dioxane (4-5 eq acid) and MeCN (0.25 volume eq to the dioxanesolution) and stirred at r.t. until LCMS indicated completedeprotection, ˜1 hr. The reaction was concentrated to dryness withtoluene (2×) (the same volume as the deprotection solution) to yield theHCl salt (f) as a white crystalline solid in approximately quantitativeyield.

The HCl salt (f) was dissolved in DMF (10-15 vol) and DIPEA (5 eq). Tothis was added the Boc-L-N-methyl Ala (1.5 eq), HOBt (1.4 eq), andEDC-HCl (1.4 eq). Coupling was complete after ˜1 hr by LCMS. Thereaction was diluted with EtOAc (15 vol) and washed with 1M HCl (2×),NaHCO₃ (1×), and brine (1×) (1:1 aq/org). The organic extract was driedover sodium sulfate and concentrated in vacuo to give the Boc-protectedconjugate product as a beige, foamy solid in ˜85% yield. TheBoc-protected conjugate was dissolved in a solution of 4M HCl/dioxane(4-5 eq acid) and MeCN (0.25 volume eq to the dioxane solution) andstirred at r.t. until LCMS indicated complete deprotection, ˜1 hr. Thereaction was concentrated to dryness with toluene (2×) (same volume asdeprotection solution) and the resulting solid was slurried in asolution of MTBE/EtOAc (70:30) (minimal volume necessary for easystirring), filtered and collected to yield crude (g) as an off-whitefree-flowing solid. The crude HCl salt (g) was suspended in MeOH (4 volminimum) and dissolved with stilling at 65° C. Warm Isopropyl Acetate(6-8 vol) is added in two portions, keeping the temperature at approx.60° C., and the solution was allowed to cool with stirring.Crystallization took place rapidly, the suspension was stirred at roomtemperature for several hours, then stirred at 0° C. for an hour beforethe solid was collected by suction filtration, washed with MeOH/iPrOAc(1:4, 2 vol) and dried to yield final the product as a white/off-whitecrystalline solid in ˜80% yield from (f).

Example 5 2-[tert-butoxycarbonyl-(1H-pyrrol-2-ylmethyl)-amino]-propionicAcid

Alanine ethyl ester (5 g, 32.5 mmol), pyrrole-2-carboxaldehyde (3.1 g,32.5 mmol), sodium cyanoborohydride (2.04 g, 32.5 mmol) and AcOH (1%)were mixed in DMF and stirred overnight. The reaction was quenched withH₂O, and DMF was evaporated. The mixture was diluted with EtOAc, washedby 0.1N NaOH, dried and concentrated to yield product 2.5 g. Theresulting ester (2.5 g, 12.8 mmol), di-tert-butyldicarbonate (3.06 g, 14mmol) were mixed in THF, H₂O with NaHCO₃ and stirred overnight. THF wasevaporated, and the mixture was diluted with EtOAc, washed by 1N NaOH,sat. NH₄Cl and brine. After dried, the mixture was concentrated to yieldthe Boc-protected ester 3.3 g. The Boc-protected ester (1.67 g, 5.6mol), lithium hydroxide mono hydrate (284 mg, 6.77 mmol) were mixed inTHF and H₂O at 0° C. THF was vacuumed off, and the solution wasacidified by dilute H₂SO₄, extracted by EtOAc twice. Organic layers werecombined, dried and evaporated.

Example 6 Tetrahydropyranylglycine

Tetrahydropyranylglycine was purchased from NovaBiochem, or synthesizedaccording to the literature: Ghosh, A. K.; Thompson, W. J.; holloway, M.K.; McKee, S. P.; Duong, T. T.; Lee, H. Y.; Munson, P. M.; Smith, A. M.;Wai, J. M; Darke, P. L.; Zugay, J. A.; Emini, E. A.; Schleife, W. A.;Huff, J. R.; Anderson, P. S. J. Med. Chem., 1993, 36, 2300-2310.

Example 7 Piperidinylglycine

Piperidinylglycine was synthesized according to the literature: Shieh,W-C.; Xue, S.; Reel, N.; Wu, R.; Fitt, J.; Repic, O. Tetrahedron:Asymmetry, 2001, 12, 2421-2425.

Example 8 4,4-difluorocyclohexylglycine

4,4-difluorocyclohexylglycine was made according to the proceduresdescribed in US 2003/0216325.

Example 9 Boc (S)-2-amino-2-(4-hydroxycyclohexyl)acetic Acid

Following the procedure of Sheih, (Tetrahedron: Asymmetry, 2001, 12,2421-2425), a solution of ketone a (8.4 g) and EtOAc (30 mL) was addedto a solution of N-Cbz-phosphonoglycine methyl ester b, TMG (4.5 mL) andEtOAc (30 mL). The solution was maintained at rt for 48 h, then washedwith 1N HCl (3×50 mL), brine (1×50 mL) dried (Na₂SO₄), filtered, andconcentrated. The residue was adsorbed onto Celite, and purified bychromatography, then further purified by re-crystallization fromEtOAc/hexanes to afford 5.2 g of product c.

Following the procedure of Sheih, (Tetrahedron: Asymmetry, 2001, 12,2421-2425), a solution of eneamide c (5.0 g), (S,S)-Me-BPE-Rh(I) (1.5 g,Strem Chemicals, Newburyport, Mass.), and MeOH (100 mL) was shakenvirgorously under 70 psi of H₂ for 48 h. The solvent was removed underreduced pressure. The residue was taken up in EtOAc, and filteredthrough SiO₂ with more EtOAc. The solvent was removed under reducedpressure to afford 4.0 g of product d as a colorless solid.

A mixture of Cbz-carbamate d, (4.0 g) Boc₂O, (2.9 g), 20% Pd(OH)₂.C (1.0g) and MeOH (30 mL) was maintained under an atmosphear of H₂ for 6 h.The mixture was filtered through Celite with MeOH. The solvent wasremoved under reduced pressure to afford 4.5 g of residue e, which wastaken on directly.

The residue e from above was dissolved in H₂O (10 mL), AcOH (30 mL), THF(5 mL), and dichloroacetic acid (3 mL) and maintained at rt overnight.Water (5 mL) was added and the solution and maintained until hydrolysiswas complete, as monitored by HPLC-MS. Solid Na₂CO₃ was added cautiouslyuntil gas evolution ceased, the mixture was diluted with aq NaHCO₃, andextracted with 10% EtOAc/DCM. The combined organic phases were washedonce with brine, dried (Na₂SO₄), filtered, and concentrated. The residuewas purified by chromatography to afford 2.9 g of product 1.

A mixture of ketone f (1.5 g) MeOH (50 ml) was treated with NaBH4 (290mg) at 0° C. for 20 min. The mixture was acidified to ˜pH1 with 10% aqcitric acid and the MeOH was removed under reduced pressure. The residuewas diluted with water and extracted with 20% EtOAc/DCM. The combinedorganic phases were washed once with brine, dried (Na₂SO₄), filtered,and concentrated. The residue was purified by chromatography to afford1.17 g of product g and 0.23 g of product h.

A mixture of ester g (1.17 g) LiOH.H2O (160 mg), THF (3 mL) and water(4.5 mL) was stirred vigorously at rt overnight. The mixture was dilutedwith brine and exhaustively extracted with EtOAc. The combined organicphases were washed once with brine, dried (Na₂SO₄), filtered, andconcentrated to afford acid i (525 mg).

Example 10 Compound 29

A mixture of amine a (1.56 mmol), 2-Bromopropionic acid (0.72 g, 4.68mmol), BOP (2.1 g, 4.68 mmol) and DIPEA (1.6 ml, 9.36 mmol) in 10 ml DMFwas stirred at room temperature for 2 hours. LCMS analysis indicatedreaction completed. 100 ml EtOAc was added to the reaction and organiclayer was washed with sat. NaHCO₃ followed by brine, dried over Na₂SO₄and concentrated to dryness. The crude material was purified bychromatography using 50% EtOAc/hexane to obtain compound b.

The compound b (0.832 g, 1.5 mmol) was treated with ethanolamine (200ul, 2.73 mmol) in 3 ml DMF and stirred overnight for completion. Thereaction mixture was purified by reverse phase HPLC to obtain twodiastereomers c (53 mg) and d (compound 29) (150 mg).

Example 11 N-Boc-N-cyclopropylmethyl-L-alanine

L-alanine methyl ester hydrochloride a (5 g, 35.8 mmol) andcyclopropanecarboxaldehyde b (2.67 ml, 35.8 mmol) were suspended in 50ml THF w/1% AcOH. Addition of 5 ml of CH₃OH made the cloudy solutionturned to clear. NaCNBH₄ (2.25 g, 35.8 mmol) was added and the reactionmixture stirred overnight. The reaction was quenched by addition of 1Naq. NaOH, extracted by EtOAc twice, organic layers were dried overNa₂SO₄ and concentrated to dryness. The crude material was purified bychromatography using 30% EtOAc/hexane (stained by ninhydrin) to obtainthe compound c (1 g, 18%). The compound c (1 g, 6.37 mmol) anddi-t-bocdicarbonate (2.1 g, 9.55 mmol) were diluted in THF (20 ml) andH₂O (20 ml), NaHCO₃ (1.3 g, 15.9 mmol) was added. The reaction mixturestirred overnight for completion. THF was removed under reducedpressure, and the aqueous layer was extracted by EtOAc 3 times. Combinedorganic layers were washed by 1N NaOH, sat, NH₄Cl followed by brine, theconcentrated to dryness. The Boc-protected compound d (1.39 g, 5.40mmol) was stirred with LiOH.H₂O (1.14 g, 27 mmol) in THF (20 ml) and H₂O(20 ml) overnight at room temperature. THF was stripped off, and theaqueous layer was adjusted to pH=4 by adding 10% citric acid, thenextracted by EtOAc 3 times. Combined organic layers were washed by brineand concentrated. The crude was purified by reverse phase C-18 columneluted by 0%-50% acetonitrile/H₂O to give pure compound e as a whitesolid (794 mg).

Example 12 Acid Fluoride Coupling Procedure

A solution of Boc-MeAla-Chg-Pro-OH (2.3 mmol) and pyridine (6.9 umol) inanhydrous dichloromethane (23 ml) was cooled to 0° C. and cyanuricfluoride (2.3 mmol) added dropwise over 30 sec. The mixture was stirredat 0° C. for 15 min, at ambient temperature for 5 hr, and then quenchedwith water. The mixture was extracted three times with dichloromethane(total 100 ml), and the combined organic phases washed with brine anddried over anhydrous sodium sulfate. Filtration and concentration invacuo yielded the peptide acid fluoride as a clear, colorless oil useddirectly without further purification.

A solution of the crude acid fluoride (0.50 mmol) and pyridine (1.5mmol) in dichloromethane (2.5 ml) was added to the solid amine (0.50mmol), and the resulting mixture stirred either at ambient temperatureor at 50° C. (sealed vessel). The mixture was poured into aqueous sodiumbicarbonate and the extracted three times with dichloromethane (total100 ml). The combined organic phases were washed with brine, dried overanhydrous sodium sulfate, filtered and concentrated in vacuo. The crudepeptide amide was used directly without further purification.

Example 13 1-phenyl-1H-pyrazol-5-amine

1-phenyl-1H-pyrazol-5-amine is commercially available from TCI America(catalog #A0174).

Example 14 3-methyl-1-phenyl-1H-pyrazol-5-amine

3-methyl-1-phenyl-1H-pyrazol-5-amine is commercially available from TCIAmerica (catalog #A1311).

Example 15 5-phenylthiazole-2,4-diamine

5-phenylthiazole-2,4-diamine is commercially available from AcrosOrganics (catalog #11234-0010).

Example 16 5-(trifluoromethyl)-4-phenylthiophen-3-amine

5-(trifluoromethyl)-4-phenylthiophen-3-amine is commercially availablefrom Acros Organics (catalog #SEW03133DA).

Example 17 4-phenyl-1H-pyrazol-3-amine

4-phenyl-1H-pyrazol-3-amine was prepared according to the proceduresdescribed in E. L. Anderson et al.; J. Med. Chem., 1964, 7, 259-268.

Example 18 5-methyl-4-phenyl-1H-pyrazol-3-amine

5-methyl-4-phenyl-1H-pyrazol-3-amine was prepared according to theprocedures described in E. L. Anderson et al.; J. Med. Chem., 1964, 7,259-268.

Example 19 3-phenyl-3H-1,2,3-triazol-4-amine

3-phenyl-3H-1,2,3-triazol-4-amine was prepared according to theprocedures described in K. M. Baines, T. W. Rourke, K. Vaughan; J. Org.Chem., 1981, 46, 856-859.

Example 20 4-phenylisoxazol-5-amine

4-phenylisoxazol-5-amine was prepared according to the proceduresdescribed in H. Peeters, W. Vogt; EP 43024.

Example 21 3-phenyl-1H-pyrazol-4-mine

3-phenyl-1H-pyrazol-4-amine was prepared according to the proceduresdescribed in C. Chen, K. Wilcoxen, J. R. McCarthy; Tetrahedron Lett.,1988, 39, 8229-8232.

Example 22 1-methyl-3-phenyl-1H-pyrazol-4-amine

1-methyl-3-phenyl-1H-pyrazol-4-amine was prepared according to theprocedures described in C. Chen, K. Wilcoxen, J. R. McCarthy;Tetrahedron Lett., 1988, 39, 8229-8232.

Example 23 1-methyl-5-phenyl-1H-pyrazol-4-amine

1-methyl-5-phenyl-1H-pyrazol-4-amine was prepared according to theprocedures described in C. Chen, K. Wilcoxen, J. R. McCarthy;Tetrahedron Lett., 1988, 39, 8229-8232.

Example 24 3-methyl-4-phenylisoxazol-5-amine

3-methyl-4-phenylisoxazol-5-amine was prepared according to theprocedures described in H. Peeters, W. Vogt; EP 43024.

Example 25 1-phenyl-1H-tetrazol-5-amine

1-phenyl-1H-tetrazol-5-amine was prepared according to the proceduresdescribed in R. A. Batey, D. A. Powell; Org. Lett., 2000, 2, 3237-3240.

Example 26 4-phenyl-1,2,5-oxadiazol-3-amine

4-phenyl-1,2,5-oxadiazol-3-amine was prepared according to theprocedures described in R. Lakhan, O. P. Singh; Ind. J. Chem., 1987,26B, 690-692.

Example 27 1-amino-5-phenyl-1H-tetrazole

1-amino-5-phenyl-1H-tetrazole was prepared according to the proceduresdescribed in T. L. Gilchrist, G. E. Gymer, C. W. Rees; J. Chem. Soc.,Perkin Trans. 1, 1975, 1747-1750.

Example 28 4-amino-3-phenyl-4H-1,2,4-triazole

4-amino-3-phenyl-4H-1,2,4-triazole was prepared according to theprocedures described in A. A. Ikizler, N. Yildirim; J. HeterocyclicChem., 1998, 35, 377-380.

Example 29 3-phenylthiophen-2-amine

3-phenylthiophen-2-amine was prepared according to the proceduresdescribed in Y. Yoshikawa et al.; EP 737682 (U.S. Pat. No. 5,747,518).

Example 30 2-phenylthiophen-3-amine

2-phenylthiophen-3-amine was prepared according to the proceduresdescribed in Y. Yoshikawa et al.; EP 737682 (U.S. Pat. No. 5,747,518).

Example 31 4-phenylthiophen-3-amine

4-phenylthiophen-3-amine was prepared according to the proceduresdescribed in G. Kirsch, D. Cagniant, P. Cagniant; J. Heterocyclic Chem.,1982, 19, 443-445.

Example 32 5-amino-4-phenylthiazole-2-thiol

5-amino-4-phenylthiazole-2-thiol was prepared according to theprocedures described in A. H. Cook, I. Heilbron, A. L. Levy; J. Chem.Soc., 1947, 1598-1609.

Example 33 2-(methylthio)-4-phenylthiazol-5-amine

2-(methylthio)-4-phenylthiazol-5-amine was prepared according to theprocedures described in A. H. Cook, I. Heilbron, A. L. Levy; J. Chem.Soc., 1947, 1598-1609.

Example 34 5-amino-2-(methylsulfinyl)-4-phenylthiazole

To 5-amino-2-(methylsulfanyl)-4-phenylthiazole (305 mg, 1.37 mmol) inacetic acid (3.0 ml) was added aqueous hydrogen peroxide (660 μl, 30%wt, 6.9 mmol) dropwise at ambient temperature. After 4 hr the mixturewas partitioned between dichloromethane (60 ml) and water (60 ml). Theorganic phase was separated, washed with brine, dried over sodiumsulfate, filtered and concentrated in vacuo. Flash chromatography onsilica gel (ethyl acetate/hexanes) yielded pure5-amino-2-(methylsulfinyl)-4-phenylthiazole (285 mg, 87%).

Example 35 5-amino-2-(methylsulfonyl)-4-phenylthiazole

To 5-amino-2-(methylsulfanyl)-4-phenylthiazole (302 mg, 1.36 mmol) indichloromethane (5.0 ml) was added portionwise 3-chloroperbenzoic acid(638 mg, 77% wt, 2.9 mmol) with cooling to 0° C. The mixture was dilutedwith dichloromethane (3.0 ml) and after 5 min allowed to warm to ambienttemperature. After 3 hr a further quantity of 3-chloroperbenzoic acid(305 mg, 77% wt, 1.4 mmol) was added portionwise. After 20 hr themixture was treated with sodium thiosulfate (2 ml, 1.0 M), poured intosaturated aqueous sodium bicarbonate and extracted three times intodichloromethane (total 100 ml). The combined organic phases were washedwith saturated aqueous sodium bicarbonate and brine, dried over sodiumsulfate and concentrated in vacuo to give a dark brown foam. Flashchromatography on silica gel (ethyl acetate/hexanes) yielded pure5-amino-2-(methylsulfonyl)-4-phenylthiazole (90 mg, 26%).

Example 36 5-amino-2-(aminosulfonyl)-4-phenylthiazole and5-amino-4-phenylthiazole

5-Amino-2-mercapto-4-phenylthiazole (1.01 g, 4.83 mmol) and phthalicanhydride (716 mg, 4.84 mmol) in acetic acid (20 ml) were heated at 100°C. for 64 hr and allowed to cool. The mixture was diluted into coldwater (150 ml) and the precipitate collected by filtration, washed withwater (50 ml) and dried under high vacuum (1.46 g, 90%). The phthalimideis contaminated with a minor quantity of disulfide but used withoutfurther purification.

2-Mercapto-4-phenyl-5-phthalimido-thiazole (203 mg, 600 μmol) in aceticacid (4.5 ml) and water (0.5 ml) at 0° C. was treated withN-chlorosuccinimide (243 mg, 1.82 mmol) in one portion. The mixture wasstirred at 0° C. for 10 min, allowed to warm to ambient temperature for1 hr and then partitioned between dichloromethane (50 ml) and water (50ml). The aqueous phase was extracted twice more with dichloromethane(2×25 ml), and the combined organic phases washed with brine and driedover sodium sulfate. Filtration and concentration in vacuo yielded amixture (231 mg) of 2-(chlorosulfonyl)-4-phenyl-5-phthalimido-thiazole(major component) with 4-phenyl-5-phthalimido-thiazole (ca. 2:1), usedwithout purification.

The crude mixture of sulfonyl chloride with4-phenyl-5-phthalimido-thiazole (231 mg) in dichloromethane (10 ml) wastreated with ammonia in methanol (900 μl, 2.0 M) dropwise at ambienttemperature. After 10 min the mixture was concentrated in vacuo. Theresidue was suspended in ethanol (10 ml), treated with ethanolichydrazine (660 μl, 1.0 M, 660 μmol) and heated to reflux. After 1.5 hr afurther portion of ethanolic hydrazine was added (660 μl, 1.0 M, 660mol) and reflux continued for 15 hr. The cooled mixture was filtered andconcentrated in vacuo. Flash chromatography on silica gel (ethylacetate/hexanes) yielded pure 5-amino-2-(aminosulfonyl)-4-phenylthiazole(56 mg, 36% for 3 steps) and 5-amino-4-phenylthiazole (17 mg, 16% for 3steps).

Example 37 5-amino-2-(tent-butylsulfanyl)-4-phenylthiazole

To a suspension of 5-amino-2-mercapto-4-phenylthiazole (210 mg, 1.01mmol) in water (1.0 ml) and tert-butanol (82 mg, 1.1 mmol) was addedconcentrated sulfuric acid (3.0 ml) with cooling to ca. 20° C. After 1.5hr at ambient temperature a further portion of tert-butanol in water(300 μl, 1.0 M, 300 μmol) was added. After 1.5 hr the mixture was pouredinto excess aqueous sodium bicarbonate and extracted three times intodichloromethane (total 120 ml). The combined organic phases were washedwith brine, dried over sodium sulfate, filtered and concentrated invacuo. Flash chromatography on silica gel (ethyl acetate/hexanes)yielded 5-amino-2-(tent-butylsulfanyl)-4-phenylthiazole (220 mg, 82%).

Example 38 5-amino-2-(tent-butylsulfinyl)-4-phenylthiazole

To 5-amino-2-(tent-butylsulfanyl)-4-phenylthiazole (102 mg, 385 mol) inacetic acid (5.0 ml) was added aqueous hydrogen peroxide (218 μl, 30%wt, 1.9 mmol) dropwise at ambient temperature. After 5 hr the mixturewas partitioned between dichloromethane (50 ml) and water (50 ml). Theaqueous phase was separated and extracted with dichloromethane (20 ml).The combined organic phases were washed with saturated aqueous sodiumbicarbonate, dried over sodium sulfate, filtered and concentrated invacuo to yield essentially pure5-amino-2-(tent-butylsulfinyl)-4-phenylthiazole (110 mg, quant.).

Example 39 5-amino-4-phenyl-2-(trifluoromethylsulfanyl)thiazole

A suspension of 5-amino-2-mercapto-4-phenylthiazole (503 mg, 2.41 mmol)in acetic acid (5.0 ml) was treated with hexane-2,5-dione (290 μl, 2.47mmol) at ambient temperature for 14 hr and then heated to reflux for 3hr. The mixture became homogeneous at reflux and on cooling deposited aprecipitate which was recovered by filtration, washed with acetic acid(3×1.0 ml) and dried in vacuo to yield the pure pyrrolidino-thiazole(624 mg, 90%) as a bright yellow microcrystalline solid.

A solution of the mercapto-thiazole (201 mg, 701 mol) and potassiumcarbonate (291 mg, 2.11 mmol) in DMF (2.0 ml) was saturated withtrifluoromethyl iodide by bubbling for 5 min, and the vessel sealed andheated at 50° C. for 30 min. The cooled mixture was again saturated withtrifluoromethyl iodide, and heated to 100° C. for 1.5 hr. The mixturewas once more saturated with trifluoromethyl iodide, returned to 100° C.(total 24 hr) and allowed to cool. The mixture was poured into water andextracted three times into ethyl acetate (total 100 ml). The combinedorganic phases were washed with water and brine, dried over magnesiumsulfate, filtered and concentrated in vacuo. Flash chromatography onsilica gel (ethyl acetate/hexanes) yielded the pure(trifluoromethylsulfanyl)-thiazole (72 mg, 29%) as a colorlesscrystalline film.

A suspension of the thiazole (72 mg) and hydroxylamine hydrochloride (71mg, 1.0 mmol) in ethanol (5.0 ml) was heated to reflux for 17 hr,diluted with acetic acid (3 ml), refluxed for a further 2 hr, andconcentrated to ca. 3 ml. The cooled mixture was treated with aqueoushydroxylamine (1.0 ml, 50% wt) and returned to reflux for 42 hr. Themixture was treated with water (50 ml) and saturated aqueous sodiumbicarbonate (50 ml) and extracted three times into dichloromethane(total 100 ml). The combined organic phases were washed with brine,dried over sodium sulfate, filtered and concentrated in vacuo. Flashchromatography on silica gel (ethyl acetate/hexanes) yielded pure5-amino-4-phenyl-2-(trifluoromethylsulfanyl)thiazole (12.5 mg, 22%).

Example 40 5-amino-4-phenyl-2-(trifluoromethyl)thiazole

α-Aminophenylacetamide (2.00 g, 13.3 mmol) in methanol (50 ml) at 0° C.was treated with ethyl trifluoroacetate (3.2 ml, 27 mmol) for 30 min andallowed to warm to ambient temperature for 18 hr. The mixture wasconcentrated in vacuo, made homogeneous with methanol and againconcentrated top yield the pure trifluoroacetamide (3.27 g, quant.).

The trifluoroacetamide (881 mg, 3.58 mmol) and Lawesson's reagent (1.45g, 3.59 mmol) were treated together with anhydrous pyridine (7.2 ml) andthe mixture heated to 100° C. for 20 hr. The cooled mixture was pouredinto saturated aqueous sodium bicarbonate and extracted three times intochloroform (total 120 ml). The combined organic phases were washed withwater containing one tenth volume saturated aqueous sodium bicarbonate,and brine, and dried over sodium sulfate. Filtration and concentrationin vacuo yielded a red-brown oil (829 mg). The crude was treated withaqueous sodium hydroxide (25 ml, 1.0 N) for 15 min and extracted threetimes into dichloromethane (total 100 ml). The combined organic phaseswere washed with aqueous sodium hydroxide (25 ml, 1.0 N) and brine,dried over sodium sulfate, filtered and concentrated in vacuo. Flashchromatography on silica gel (ethyl acetate/hexanes) yielded pure5-amino-4-phenyl-2-(trifluoromethyl)thiazole (65 mg, 7.5%).

Example 41 3-amino-4-phenyl-1,2,5-thiadiazole

To a solution of sulfur monochloride (24.0 g, 178 mmol) in DMF (30 ml)at 0° C. was added α-aminophenylacetonitrile hydrochloride (10.0 g, 59.3mmol) portionwise over 20 min. After 40 min the mixture was allowed towarm to ambient temperature for 20 min, diluted with DMF (20 ml) andstirred for a further 20 hr before pouring into ice-water. The mixturewas extracted with ether (200 ml), filtered, and the extracted twicemore with ether (2×50 ml). The combined organic phases were washed withbrine, dried over magnesium sulfate and concentrated in vacuo to give3-chloro-4-phenyl-1,2,5-thiadiazole as a mobile orange oil (10.1 g,87%). Short-path distillation of this oil (9.35 g) at reduced pressureyielded a clear, colorless oil (7.75 g, 83%) which crystallized onstanding.

3-Chloro-4-phenyl-1,2,5-thiadiazole (3.19 g, 16.2 mmol) in THF (32 ml)at 0° C. was treated dropwise with a solution of lithiumbis(trimethylsilyl)amide in THF (17.0 ml, 1.0 M, 17.0 mmol). After 10min the mixture allowed to warm to ambient temperature for 1.5 hr,treated with 1N hydrochloric acid, and extracted three times into ether(total 300 ml). The combined organic phases were washed with saturatedaqueous sodium bicarbonate and brine, and dried over magnesium sulfateand concentrated in vacuo. The residue was dissolved in methanol (50 ml)and triethylamine (0.5 ml) was heated to reflux for 15 hr and againconcentrated in vacuo. Flash chromatography on silica gel (ethylacetate/hexanes) yielded 3-amino-4-phenyl-1,2,5-thiadiazole (1.96 g,68%) as a colorless solid.

Example 42 5-amino-2-methyl-4-phenylthiazole

To a suspension of α-aminophenylacetonitrile hydrochloride (3.37 g, 20.0mmol) and powdered sulfur (641 mg, 20.0 mmol) in ethanol (20 ml) at 0°C. was added triethylamine (4.18 ml, 30.0 mmol) and then acetaldehyde(2.3 ml, 41 mmol). The vessel was sealed and heated to 60-70° C. for 1hr. The cooled mixture was filtered and concentrated in vacuo, and theresidue treated with ethanol (20 ml) and hydrochloric acid (20 ml, 1N)for 15 hr. The mixture was treated with aqueous sodium carbonate andextracted three times into ethyl acetate (total 300 ml). The combinedorganic phases were washed with brine, dried over sodium sulfate andconcentrated in vacuo to give a dark brown oil. Flash chromatography onsilica gel (ethyl acetate/hexanes) yielded5-amino-2-methyl-4-phenylthiazole (1.31 g, 34%), which crystallized fromtoluene.

Example 43 5-amino-2-methyl-4-phenylthiazole

A suspension of α-aminophenylacetonitrile hydrochloride (1.69 g, 10.0mmol), powdered sulfur (321 mg, 10.0 mmol) and 4-pyridinecarboxaldehyde(1.91 ml, 20.0 mmol) in ethanol (10 ml) was treated with triethylamine(2.09 ml, 15.0 mmol), and the mixture stirred at 50° C. for 80 min. Thecooled mixture was diluted with ethanol (5 ml) and treated with aqueoushydroxylamine (700 μl, 50% wt, 11 mmol) at ambient temperature for 15hr, and diluted with dichloromethane (50 ml). Saturated aqueous sodiumbicarbonate was added and the separated aqueous phase was extractedtwice more with dichloromethane (total 100 ml). The combined organicphases were dried over sodium sulfate and concentrated in vacuo to givea dark brown oily foam (3.23 g). Flash chromatography on silica gel(ethyl acetate/hexanes) yielded 5-amino-2-(4-pyridyl)-4-phenylthiazole(1.41 g, 56%).

Example 44 2,4-diphenylthiazol-5-amine

2,4-diphenylthiazol-5-amine was prepared according to the proceduresdescribed in K. Gewald, H. Schonfelder, U. Hain; J. Prakt. Chem., 1974,361, 299-303.

Example 45 4-phenyl-2-(pyridin-2-yl)thiazol-5-amine

4-phenyl-2-(pyridin-2-yl)thiazol-5-amine was prepared according to theprocedures described in K. Gewald, H. Schonfelder, U. Hain; J. Prakt.Chem., 1974, 361, 299-303

Example 46 4-phenyl-2-(pyridin-3-yl)thiazol-5-amine

4-phenyl-2-(pyridin-3-yl)thiazol-5-amine was prepared according to theprocedures described in K. Gewald, H. Schonfelder, U. Hain; J. Prakt.Chem., 1974, 361, 299-303

Example 47 5-amino-2-(Fmoc-amino)-4-phenylthiazole

A suspension of α-aminophenylacetonitrile hydrochloride (3.19 g, 18.9mmol) and Fmoc-isothiocyanate (5.31 g, 18.9 mmol) in DCM was treatedwith ethyldiisopropylamine (3.62 ml, 20.8 mmol) at 0° C. for 1 hr andthen at ambient temperature for 3 hr. The mixture was poured intosaturated aqueous sodium bicarbonate and extracted three times intoethyl acetate. The combined organic phases were washed with water andbrine, and dried over sodium sulfate and concentrated in vacuo. Flashchromatography on silica gel (ethyl acetate/hexanes) yielded5-amino-2-(Fmoc-amino)-4-phenylthiazole (3.75 g, 48%).

Example 48 N-(5-amino-4-phenylthiazol-2-yl)acetamide

N-(5-amino-4-phenylthiazol-2-yl)acetamide was prepared according toprocedures similar to those described in example 47.

Example 49 N-(5-amino-4-phenylthiazol-2-yl)benzamide

N-(5-amino-4-phenylthiazol-2-yl)benzamide was prepared according toprocedures similar to those described in example 47.

Example 50 Ethyl 5-amino-4-phenylthiazol-2-ylcarbamate

ethyl 5-amino-4-phenylthiazol-2-ylcarbamate was prepared according toprocedures similar to those described in example 47.

Example 51 N-(5-amino-4-(2-chlorophenyl)thiazol-2-yl)acetamide

N-(5-amino-4-(2-chlorophenyl)thiazol-2-yl)acetamide was preparedaccording to procedures similar to those described in example 47.

Example 52 (9H-fluoren-9-yl)methyl5-amino-4-(2-chlorophenyl)thiazol-2-ylcarbamate

(9H-fluoren-9-yl)methyl 5-amino-4-(2-chlorophenyl)thiazol-2-ylcarbamatewas prepared according to procedures similar to those described inexample 47.

Example 53 5-amino-2-(1-imidazolyl)-4-phenylthiazole

A suspension of α-aminophenylacetonitrile hydrochloride (5.01 g, 29.7mmol) and thiocarbonyl diimidazole (5.30 g, 29.7 mmol) in DCM (100 ml)was treated with ethyldiisopropylamine (5.69 ml, 32.7 mmol) at 0° C. for15 min and then at ambient temperature for 3 hr. The mixture was pouredinto saturated aqueous sodium bicarbonate (50 ml) and water (150 ml),and extracted three times into dichloromethane (total 300 ml). Thecombined organic phases were washed with brine, dried over magnesiumsulfate and concentrated in vacuo to give a dark brown oil (8.18 g).Flash chromatography on silica gel (ethyl acetate/hexanes) yielded5-amino-2-(1-imidazolyl)-4-phenylthiazole (2.47 g, 34%).

Example 54 MeAla-Chg-Pro peptide amide of 2,5-diamino-4-phenylthiazole

5-Amino-2-(Fmoc-amino)-4-phenylthiazole (250 mg, 605 μmol) was treatedwith the acid fluoride

(730 μmol; derived form Boc-MeAla-Chg-Pro-OH as previously described)and pyridine (147 μl, 1.82 mmol) in dichloromethane (2.0 ml) at ambienttemperature for 6 days. The mixture was poured into saturated aqueoussodium bicarbonate and extracted three times into dichloromethane (total100 ml). The combined organic phases were washed with brine, dried overmagnesium sulfate and concentrated in vacuo to yield the crude peptideamide as a yellow oil (525 mg), used subsequently without purification.

The crude peptide amide in DMF (9.0 ml) was treated with piperidine (1.0ml) at ambient temperature for 20 min and then concentrated in vacuo.Flash chromatography on silica gel (ethyl acetate/hexanes) yielded the2,5-diamino-4-phenylthiazole peptide amide (228 mg, 61% for 2 steps).

The crude peptide amide (48 mg, 78 μmol) in dichloromethane (2.0 ml) wastreated with trifluoroacetic acid (2.0 ml) at ambient temperature for 30min. The mixture was concentrated in vacuo, made homogeneous withdichloromethane and again concentrated. The residue was purified bypreparative reverse phase HPLC (acetonitrile/water) to yield the fullydeprotected peptide amide trifluoroacetic acid salt (42 mg, 73%) as awhite amorphous solid.

Example 55 MeAla-Chg-Pro peptide amide of2,5-diamino-4-(3-chlorophenyl)thiazole

MeAla-Chg-Pro peptide amide of 2,5-diamino-4-(3-chlorophenyl)thiazolewas prepared using the same procedures described in example 55.

Example 56 MeAla-Chg-Pro amide of5-amino-2-(pivaloylamino)-4-phenylthiazole

The Boc-peptide amino-thiazole (48 mg, 78 μmol) andethyldiisopropylamine (140 μl, 0.80 mmol) in dichloromethane (2.0 ml)were treated with pivaloyl chloride (50 μl, 0.40 mmol) at ambienttemperature for 3 hr, and then with saturated aqueous sodium bicarbonateand extracted three times into dichloromethane (total 60 ml). Thecombined organic phases were washed with brine, dried over magnesiumsulfate and concentrated in vacuo. The crude oil was treated withtrifluoroacetic acid (5.0 ml) in dichloromethane (5.0 ml) at ambienttemperature for 20 min. The mixture was concentrated in vacuo, madehomogeneous with dichloromethane and again concentrated. The residue wasdissolved in aqueous acetic acid (50%) for purification by preparativereverse phase HPLC (acetonitrile/water) to yield the pure peptide amidetrifluoroacetic acid salt (38 mg, 68% for 2 steps) as a white amorphoussolid.

Example 57 MeAla-Chg-Pro amide of5-amino-2-(pivaloylamino)-4-phenylthiazole

The Boc-peptide amino-thiazole (38 mg, 62 μmol) andethyldiisopropylamine (107 μl, 0.61 mmol) in dichloromethane (2.0 ml)were treated with methanesulfonyl chloride (24 μl, 0.31 mmol) at ambienttemperature for 20 min, and then with saturated aqueous sodiumbicarbonate and extracted three times into dichloromethane. The combinedorganic phases were washed with brine, dried over magnesium sulfate andconcentrated in vacuo. The crude oil was treated with trifluoroaceticacid (4 ml) in dichloromethane (4 ml) at ambient temperature for 20 min.The mixture was concentrated in vacuo, made homogeneous withdichloromethane and again concentrated. The residue was dissolved inaqueous acetic acid (50%) for purification by preparative reverse phaseHPLC (acetonitrile/water) to yield the pure peptide amidetrifluoroacetic acid salt (11 mg, 23% for 2 steps) as a white amorphoussolid.

Example 57 2-(acetylamino)-4-amino-5-phenylthiazole

α-Bromophenylacetonitrile (1.08 g, 5.48 mmol) in ethanol (10 ml) wastreated with N-acetylthiourea (649 mg, 5.49 mmol) at ambient temperaturefor 4 hr, and then heated to reflux for 3.5 hr. The cooled mixture wasconcentrated in vacuo and then partitioned between dichloromethane andsaturated aqueous sodium bicarbonate. The organic phase was washed withbrine, dried over sodium sulfate, filtered and concentrated in vacuo.Flash chromatography on silica gel (ethyl acetate/hexanes) yielded2-(acetylamino)-4-amino-5-phenylthiazole (295 mg, 23%).

Example 58 2,5-diphenylthiazol-4-amine

2,5-diphenylthiazol-4-amine was prepared using the same proceduresdescribed in example 57.

Example 59 5-phenyl-2-(pyrazin-2-yl)thiazol-4-amine

5-phenyl-2-(pyrazin-2-yl)thiazol-4-amine was prepared using the sameprocedures described in example 57.

Example 60 5-amino-1-(3′-nitrophenyl)pyrazole

3-Nitrophenylhydrazine hydrochloride (7.03 g, 36.3 mmol),diisopropylethylamine (9.5 ml, 54.5 mmol), and ethanol (60 ml) werestirred under nitrogen at room temperature for 2 h.Ethoxymethylenemalononitrile (4.52 g, 36.3 mmol) was added, after whichthe reaction was refluxed for 1 h. Reaction was cooled to roomtemperature. Solvent was removed under reduced pressure untilprecipitate crashed out. The solid was filtered to yield 6.54 g of thecyclized product (78% yield).

5-amino-1-(3′-nitrophenyl)-4-cyanopyrazole (559 mg, 2.44 mmol) andphosphoric acid (86%, 6 ml) were refluxed at 170° C. for 15 h. Thereaction was cooled to room temperature and neutralized with ammoniumhydroxide. The organics were extracted three times with diethyl ether(total 40 ml), washed with brine, and dried over magnesium sulfate.Removal of solvent gave 5-amino-1-(3′-nitrophenyl)-pyrazole as a yellowpowder (398 mg, 80% yield).

Example 61 1-(2-fluorophenyl)-1H-pyrazol-5-amine

1-(2-fluorophenyl)-1H-pyrazol-5-amine was prepared using the sameprocedures described in example 60.

Example 62 1-(3-chlorophenyl)-1H-pyrazol-5-amine

1-(3-chlorophenyl)-1H-pyrazol-5-amine was prepared using the sameprocedures described in example 60.

Example 63 1-(3-fluorophenyl)-1H-pyrazol-5-amine

1-(3-fluorophenyl)-1H-pyrazol-5-amine was prepared using the sameprocedures described in example 60.

Example 64 1-(3-bromophenyl)-1H-pyrazol-5-amine

1-(3-bromophenyl)-1H-pyrazol-5-amine was prepared using the sameprocedures described in example 60.

Example 65 1-(3-trichloromethylphenyl)-1H-pyrazol-5-amine

1-(3-trichloromethylphenyl)-1H-pyrazol-5-amine was prepared using thesame procedures described in example 60.

Example 66 1-(pyridin-2-yl)-1H-pyrazol-5-amine

1-(pyridin-2-yl)-1H-pyrazol-5-amine was prepared using the sameprocedures described in example 60.

Example 67 1-(3-methoxyphenyl)-1H-pyrazol-5-amine

1-(3-methoxyphenyl)-1H-pyrazol-5-amine was isolated followingdecyanation of 5-amino-4-cyano-1-(3′-methoxyphenyl)pyrazole in example60.

Example 67 1-(3-hydroxyphenyl)-1H-pyrazol-5-amine

1-(3-hydroxyphenyl)-1H-pyrazol-5-amine was prepared using the sameprocedures described in example 60.

Example 68 4-amino-5-phenyl-1,2,3-thiadiazole

Phenylpyruvic acid (25 g, 149 mmol) and ethyl carbazate (16 g, 149 mmol)were refluxed in benzene (225 ml) for 2 hr, and the mixture concentratedin vacuo. The crude was dissolved in minimum warm dichloromethane toyield the hydrazone as a yellow precipitate upon cooling to ambienttemperature, isolated by filtration (30.4 g, 81%) and used withoutfurther purification.

Diazomethane was generated by adding a solution of Diazald(N-methyl-N-nitroso-p-toluenesulfonamide; 18.6 g, 86.9 mmol) in diethylether (180 ml) to a solution of potassium hydroxide (18.2 g, 325 mmol)in water (37 ml) and 2-(2-ethoxyethoxy)-ethanol (37 ml) at 65° C.,dropwise over 45 min. Distillation thus produced an ethereal solution ofdiazomethane which was added directly to a stirred solution of thehydrazone (10.9 g, 43.5 mmol) in methanol (150 ml) at 0° C. The systemwas rinsed with excess diethyl ether until distillate became clear, themixture treated with acetic acid (1 ml), and concentrated in vacuo. Theresulting oil was partitioned between ethyl acetate (200 ml) and sodiumbicarbonate (200 ml), and the organic phase dried over sodium sulfate.Filtration and concentration in vacuo yielded the methyl ester as ayellow solid (10.2 g, 89%).

The hydrazone-methyl ester (10.2 g, 38.6 mmol) was treated with thionylchloride (25 ml, 343 mmol) at ambient temperature for 24 hr, and themixture concentrated in vacuo. Crystallization from hexanes yielded thethiadiazole-methyl ester (4.81 g, 56%).

The thiadiazole-methyl ester (2.79 g, 12.7 mmol) was treated withhydrazine hydrate (1.09 ml, 93.9 mmol) in methanol (50 ml) at ambienttemperature for 24 hr, and the resulting white precipitate recovered byfiltration. Recrystallization from isopropanol yielded thethiadiazole-hydrazide (3.99 g, 83%).

The thiadiazole-hydrazide (3.99 g, 18.1 mmol) in water (40 ml) andconcentrated hydrochloric acid (1.8 ml, 21.9 mmol) was treated dropwisewith a solution of sodium nitrite (1.52 g, 21.3 mmol) in water (15 ml)at 0° C. for 2 hr. The resulting precipitate was recovered by filtrationto yield the thiadiazole-acid azide as an off-white solid (3.95 g, 94%).

According to the procedures described in K. Masuda et al.; Chem. Pharm.Bull., 1981, 29, 1743-1747, the thiadiazole-acid azide (3.95 g, 17.1mmol) was at reflux in ethanol (40 ml) for 45 min, and the mixtureconcentrated in vacuo. Crystallization from benzene yielded the ethylcarbamate (3.37 g, 74%).

The ethyl carbamate (399 mg, 1.60 mmol) and hydrogen bromide in aceticacid (3 ml, 30% wt) were heated in a sealed vessel at 80° C. for 18 hr.The cooled mixture was partitioned between ethyl acetate (15 ml) andwater (15 ml), and the organic phase concentrated in vacuo. Flashchromatography on silica gel (ethyl acetate/hexanes) yielded4-amino-5-phenyl-1,2,3-thiadiazole (136 mg, 49%).

Example 69 4-amino-5-phenylisoxazole

5-Phenyl-4-isoxazolecarboxylic acid (460 mg, 2.36 mmol) and thionylchloride (1.71 ml, 23.6 mmol) were heated at reflux for 3 hr, and themixture concentrated in vacuo to yield the acid chloride which was usedwithout purification.

The crude acid chloride in acetone (7 ml) was treated with a solution ofsodium azide (165 mg, 2.62 mmol) in water (2 ml) at 0° C. for 1.5 hr,and allowed to warm to ambient temperature and concentrated in vacuo.The resulting white solid was washed with water and dried in vacuo, andused without purification.

The acid azide (409 mg, 1.91 mmol) was heated at reflux in methanol for6 hr, and the mixture concentrated in vacuo to yield the methylcarbamate as a white solid, used without purification.

The methyl carbamate (378 mg, 1.73 mmol) was treated with hydrobromicacid (13 ml, 48% wt, 115 mmol), made homogeneous with acetic acid (2ml), and heated at 65° C. for 48 hr, and allowed to cool. The mixturewas neutralized with aqueous sodium hydroxide and extracted with ethylacetate (2×125 ml). The combined organic phases were dried over sodiumsulfate and concentrated in vacuo to yield 4-amino-5-phenylisoxazole asa white solid (193 mg, 70%).

Example 70 Synthesis of 5-alkyl-2-amino-3-phenylthiophenes

Benzyl cyanide (2.33 ml, 20 mmol) was treated with Verkade's base(2,8,9-trimethyl-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane; 441mg, 2.0 mmol) and 3,3-dimethylbutyraldehyde (2.64 ml, 200 mmol) inmethanol (4 ml) and the mixture heated in a sealed vessel at 45° C. for16 hr. The cooled mixture was concentrated in vacuo to yield theunsaturated nitrile as a colorless oil, used without purification.

The nitrile (10.0 mmol), potassium carbonate (2.34 g, 23.4 mmol) andpowdered sulfur (330 mg, 10.3 mmol) in ethanol (2 ml) were heated in asealed vessel at 160° C. for 24 hr. The cooled mixture was diluted withwater, extracted twice into diethyl ether and the combined organicsconcentrated in vacuo. Flash chromatography on silica gel (ethylacetate/hexanes) yielded 5-amino-2-tert-butyl-4-phenylthiazole (75%).

Example 71 5-methyl-3-phenylthiophen-2-amine

5-methyl-3-phenylthiophen-2-amine was prepared using the same proceduresdescribed in example 70.

Example 72 5-isopropyl-3-phenylthiophen-2-amine

5-isopropyl-3-phenylthiophen-2-amine was prepared using the sameprocedures described in example 70.

Example 73 2-amino-5-chloro-3-phenylthiophene

2-Amino-3-phenyl-thiophene (12.0 mmol) in THF (7 ml) was treated withdi-tent-butyl dicarbonate (2.97 g, 13.3 mmol) and diisopropylethylamine(3.15 ml, 18.1 mmol) at ambient temperature for 60 hr, and the mixtureconcentrated in vacuo. Flash chromatography on silica gel (ethylacetate/hexanes) yielded 2-(N-Boc-amino)-3-phenyl-thiophene (1.98 g,59%).

To 2-(N-Boc-amino)-3-phenyl-thiophene (89 mg, 0.32 mmol) indichloromethane (4 ml) at 0° C. was slowly added N-chlorosuccinimide (48mg, 0.36 mmol), and the mixture allowed warm to ambient temperature for16 hr. The mixture was diluted with dichloromethane, washed with water,and the organic phase concentrated in vacuo. Flash chromatography onsilica gel (ethyl acetate/hexanes) yielded2-(N-Boc-amino)-5-chloro-3-phenyl-thiophene (66 mg, 66%).

2-(N-Boc-amino)-5-chloro-3-phenyl-thiophene (66 mg, 0.21 mmol) wastreated with trifluoroacetic acid (1 ml) in dichloromethane (3 ml) atambient temperature for 1 hr. The mixture was diluted with DMF (1 ml)and the more volatile materials removed under reduced pressure. Theresulting DMF solution of 2-amino-5-chloro-3-phenylthiophene was used inthe subsequent coupling step without purification.

Example 74 1-Methyl-4-(methylamino)-3-phenylpyrazole

To 1-methyl-4-amino-3-phenylpyrazole (572 mg, 3.30 mmol) anddi-tert-butyl dicarbonate (799 mg, 3.66 mmol) in THF (10 ml) and water(3 ml) was added dropwise saturated aqueous sodium bicarbonate (3 ml,1.2 M, 3.6 mmol). The mixture was stirred at ambient temperature for 7hr and then poured into aqueous citric acid (0.5 M) and extracted threetimes into ether (total 100 ml). The combined organic phases were washedwith saturated aqueous sodium bicarbonate and brine, dried overmagnesium sulfate and concentrated in vacuo to yield the crude carbamateas a brown oil (920 mg), used subsequently without purification.

A suspension of sodium hydride in mineral oil (327 mg, 60% wt, 8.18mmol) was washed with THF (2×5 ml) and suspended in THF (3.0 ml) at 0°C. To this was added dropwise the pyrazole (744 mg, 2.72 mmol) in THF(5.0 ml), and after 15 min, methyl iodide (187 μl, 3.00 mmol). After afurther 30 min at 0° C. the mixture was allowed to warm to ambienttemperature for 18 hr and then treated with saturated aqueous ammoniumchloride and sufficient water to dissolve solids. The mixture wasextracted three times into ether (total 120 ml), and the combinedorganic phases washed with brine, dried over magnesium sulfate andconcentrated in vacuo to yield the crude N-methyl carbamate as a amberoil (750 mg, 96%), used without purification.

The crude N-methyl carbamate in DCM (1.0 ml) was treated withtrifluoroacetic acid (1.0 ml) at ambient temperature for 40 min. Themixture was concentrated in vacuo, made homogeneous with dichloromethaneand again concentrated to yield essentially pure1-methyl-4-(methylamino)-3-phenylpyrazole (150 mg, quant.) as a brownoil.

Example 75 N-methyl-4-phenyl-1,2,3-thiadiazol-5-amine

N-methyl-4-phenyl-1,2,3-thiadiazol-5-amine was prepared using the sameprocedures described in example 74.

Example 76 1-tent-Butyl-4-amino-3-phenylpyrazole and1-tent-butyl-4-amino-5-phenylpyrazole

A solution of 2-bromoacetophenone (30.0 g, 151 mmol) in DMF (120 ml) wastreated with potassium phthalimide (30.8 g, 166 mmol) portionwise atambient temperature, and then heated to 40° C. for 3.5 hr. The cooledmixture was poured into water (600 ml) and extracted with chloroform(300 ml then 100 ml). The combined organic phases were washed withsodium hydroxide (200 ml, 0.2 N), water (2×100 ml) and brine (100 ml),dried over magnesium sulfate and concentrated in vacuo. The resultingcream solid was suspended in ether (100 ml), recovered by filtration,washed with ether (100 ml) and dried in vacuo to yield pure2-phthalimido-acetophenone as a white solid (34.3 g, 86%).

According to the procedures described in C. Chen, K. Wilcoxen, J. R.McCarthy; Tetrahedron Lett., 1988, 39, 8229-8232 a suspension of2-phthalimidoacetophenone (13.3 g, 50.0 mmol) in dimethylformamidedimethyl acetal (26.7 ml, 200 mmol) was heated at reflux for 28 hr andconcentrated in vacuo. The resulting amber oil was crystallized formisopropanol (100 ml) and washed with isopropanol (2×5 ml) to yield of3-(dimethylamino)-1-phenyl-2-phthalimido-2-propen-1-one as yellowneedles (13.7 g, 85%).

A mixture of 3-(dimethylamino)-1-phenyl-2-phthalimido-2-propen-1-one(3.00 g, 9.38 mmol) and tert-butyl hydrazine hydrochloride (1.29 g, 10.3mmol) in ethanol (94 ml) and water (9.4 ml) was stirred at ambienttemperature for 64 hr and then heated at reflux for 24 hr. The cooledmixture was treated with hydrazine (590 μl, 18.8 mmol) and returned toreflux for 75 min. On cooling and standing at ambient temperature aprecipitate formed. The mixture was filtered, the solid washed with amixture of ethanol (5 ml) and water (0.5 ml), and the filtrateconcentrated in vacuo. The residue was partitioned between ether (250ml) and saturated aqueous sodium bicarbonate (50 ml) diluted with water(100 ml), and the aqueous phase extracted twice more with ether (2×50ml). The combined organic phases were washed with brine, dried oversodium sulfate and concentrated in vacuo to give a pale solid (1.92 g).Flash chromatography on silica gel (ethyl acetate/hexanes) yielded1-tent-butyl-4-amino-3-phenylpyrazole (1.52 g, 75% for 2 steps) and1-tent-butyl-4-amino-5-phenylpyrazole (114 mg, 6% for 2 steps).

Example 77 1-(2,2,2-trifluoroethyl)-3-phenyl-1H-pyrazol-4-amine and1-(2,2,2-trifluoroethyl)-5-phenyl-1H-pyrazol-4-amine

1-(2,2,2-trifluoroethyl)-3-phenyl-1H-pyrazol-4-amine and1-(2,2,2-trifluoroethyl)-5-phenyl-1H-pyrazol-4-amine were preparedsimilarly from 2,2,2-trifluoroethylhydrazine according to the proceduresdescribed in example 76.

Example 78 IAP Inhibition Assays

In the following experiments was used a chimeric BIR domain referred toas MLXBIR3SG in which 11 of 110 residues correspond to those found inXIAP-BIR3, while the remainder correspond to ML-IAP-BIR. The chimericprotein MLXBIR3SG was shown to bind and inhibit caspase-9 significantlybetter than either of the native BIR domains, but bound Smac-basedpeptides and mature Smac with affinities similar to those of nativeML-IAP-BIR. The improved caspase-9 inhibition of the chimeric BIR domainMLXBIR3SG has been correlated with increased inhibition ofdoxorubicin-induced apoptosis when transfected into MCF7 cells.

MLXBIR3SG sequence: (SEQ ID NO.: 1)MGSSHHHHHHSSGLVPRGSHMLETEEEEEEGAGATLSRGPAFPGMGSEELRLASFYDWPLTAEVPPELLAAAGFFHTGHQDKVRCFFCYGGLQSWKRGDDPWTEHAKWFPGCQFLLRSKGQEYINNIHLTHSLTR-FRET Peptide Binding Assay

Time-Resolved Fluorescence Resonance Energy Transfer competitionexperiments were performed on the Wallac Victor2 Multilabeled CounterReader (Perkin Elmer Life and Analytical Sciences, Inc.) according tothe procedures of Kolb et al (Journal of Biomolecular Screening, 1996,1(4):203). A reagent cocktail containing 300 nM his-tagged MLXBIR3SG;200 nM biotinylated SMAC peptide (AVPI); 5 μg/mL anti-hisallophycocyanin (XL665) (CISBio International); and 200 ng/mLstreptavidin-europium (Perkin Elmer) was prepared in reagent buffer (50mM Tris [pH 7.2], 120 mM NaCl, 0.1% bovine globulins, 5 mM DTT and 0.05%octylglucoside). (Alternatively, this cocktail can be made usingeuropium-labeled anti-His (Perkin Elmer) andstreptavidin-allophycocyanin (Perkin Elmer) at concentrations of 6.5 nMand 25 nM, respectively). The reagent cocktail was incubated at roomtemperature for 30 minutes. After incubation, the cocktail was added to1:3 serial dilutions of an antagonist compound (starting concentrationof 50 μM) in 384-well black FIA plates (Greiner Bio-One, Inc.). After a90 minute incubation at room temperature, the fluorescence was read withfilters for the excitation of europium (340 nm) and for the emissionwavelengths of europium (615 nm) and a allophycocyanin (665 nm).Antagonist data were calculated as a ratio of the emission signal ofallophycocyanin at 665 nm to that of the emission of europium at 615 nm(these ratios were multiplied by a factor of 10,000 for ease of datamanipulation). The resulting values were plotted as a function ofantagonist concentration and fit to a 4-parameter equation usingKaleidograph software (Synergy Software, Reading, Pa.). Indications ofantagonist potency were determined from the IC50 values. Compounds ofthe invention where found to have IAP inhibitory activity which wasdemonstrated in this assay.

Fluorescence Polarization Peptide Binding Assay

Polarization experiments were performed on an Analyst HT 96-384(Molecular Devices Corp.) according to the procedure of Keating, S. M.,Marsters, J, Beresini, M., Ladner, C., Zioncheck, K., Clark, K.,Arellano, F., and Bodary., S. (2000) in Proceedings of SPIE: In VitroDiagnostic Instrumentation (Cohn, G. E., Ed.) pp 128-137, Bellingham,Wash. Samples for fluorescence polarization affinity measurements wereprepared by addition of 1:2 serial dilutions starting at a finalconcentration of 5 μM of MLXBIR3SG in polarization buffer (50 mM Tris[pH 7.2], 120 mM NaCl, 1% bovine globulins 5 mM DTT and 0.05%octylglucoside) to 5-carboxyflourescein-conjugated AVPdi-Phe-NH₂(AVP-diPhe-FAM) at 5 nM final concentration.

The reactions were read after an incubation time of 10 minutes at roomtemperature with standard cut-off filters for the fluoresceinfluorophore (λ_(ex)=485 nm; λ_(em)=530 nm) in 96-well black HE96 plates(Molecular Devices Corp.). Fluorescence values were plotted as afunction of the protein concentration, and the IC50s were obtained byfitting the data to a 4-parameter equation using Kaleidograph software(Synergy software, Reading, Pa.). Competition experiments were performedby addition of the MLXBIR3SG at 30 nM to wells containing 5 nM of theAVP-diPhe-FAM probe as well as 1:3 serial dilutions of antagonistcompounds starting at a concentration of 300 μM in the polarizationbuffer. Samples were read after a 10-minute incubation. Fluorescencepolarization values were plotted as a function of the antagonistconcentration, and the IC₅₀ values were obtained by fitting the data toa 4-parameter equation using Kaleidograph software (Synergy software,Reading, Pa.). Inhibition constants (K_(i)) for the antagonists weredetermined from the IC₅₀ values. Compounds of the invention where foundto have IAP inhibitory activity which was demonstrated in this assay.

1. A method of inducing apoptosis in a cell comprising introducing intosaid cell a compound a therapeutically effective amount of a compound offormula I:

wherein X₁, X₂ and X₃ are independently O or S; Y is (CHR₇)_(n), O or S;wherein n is 1 or 2 and R₇ is H, halogen, alkyl, aryl, aralkyl, amino,arylamino, alkylamino, aralkylamino, alkoxy, aryloxy or aralkyloxy, A isa 5-member heterocycle comprising 1 to 4 heteroatoms optionallysubstituted with amino, hydroxyl, mercapto, halogen, carboxyl, amidino,guanidino, alkyl, alkoxy, aryl, aryloxy, acyl, acyloxy, acylamino,alkoxycarbonylamino, cycloalkyl, alkylthio, alkylsulfinyl,alkylsulfonyl, aminosulfonyl, alkylaminosulfonyl, alkylsulfonylamino ora heterocycle; wherein each alkyl, alkoxy, aryl, aryloxy, acyl, acyloxy,acylamino, cycloalkyl and heterocycle substitution is optionallysubstituted with hydroxyl, halogen, mercapto, carboxyl, alkyl, alkoxy,haloalkyl, amino, nitro, cyano, cycloalkyl, aryl or a heterocycle; R₁ isH or R₁ and R₂ together form a 5-8 member ring; R₂ is alkyl, cycloalkyl,cycloalkylalkyl, aryl, aralkyl, a heterocycle or heterocyclylalkyl; eachoptionally substituted with hydroxyl, mercapto, halogen, amino,carboxyl, alkyl, haloalkyl, alkoxy or alkylthio; R₃ is H or alkyl; R₄and R₄′ are independently H, hydroxyl, amino, alkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein eachalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl andheteroarylalkyl is optionally substituted with halogen, hydroxyl,mercapto, carboxyl, alkyl, alkoxy, amino and nitro; R₅ and R₅′ are eachindependently H or alkyl; R₆, and R₆′ are each independently H, alkyl,aryl or aralkyl; and a pharmaceutically acceptable salt thereof.
 2. Amethod of sensitizing a cell to an apoptotic signal comprisingintroducing into said cell a compound a therapeutically effective amountof a compound of formula I:

wherein X₁, X₂ and X₃ are independently O or S; Y is (CHR₇)_(n), O or S;wherein n is 1 or 2 and R₇ is H, halogen, alkyl, aryl, aralkyl, amino,arylamino, alkylamino, aralkylamino, alkoxy, aryloxy or aralkyloxy; A isa 5-member heterocycle comprising 1 to 4 heteroatoms optionallysubstituted with amino, hydroxyl, mercapto, halogen, carboxyl, amidino,guanidino, alkyl, alkoxy, aryl, aryloxy, acyl, acyloxy, acylamino,alkoxycarbonylamino, cycloalkyl, alkylthio, alkylsulfinyl,alkylsulfonyl, aminosulfonyl, alkylaminosulfonyl, alkylsulfonylamino ora heterocycle; wherein each alkyl, alkoxy, aryl, aryloxy, acyl, acyloxy,acylamino, cycloalkyl and heterocycle substitution is optionallysubstituted with hydroxyl, halogen, mercapto, carboxyl, alkyl, alkoxy,haloalkyl, amino, nitro, cyano, cycloalkyl, aryl or a heterocycle; R₁ isH or R₁ and R₂ together form a 5-8 member ring; R₂ is alkyl, cycloalkyl,cycloalkylalkyl, aryl, aralkyl, a heterocycle or heterocyclylalkyl; eachoptionally substituted with hydroxyl, mercapto, halogen, amino,carboxyl, alkyl, haloalkyl, alkoxy or alkylthio; R₃ is H or alkyl; R₄and R₄′ are independently H, hydroxyl, amino, alkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroarylalkyl wherein eachalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl andheteroarylalkyl is optionally substituted with halogen, hydroxyl,mercapto, carboxyl, alkyl, alkoxy, amino and nitro; R₅, and R₅′ are eachindependently H or alkyl; R₆, and R₆′ are each independently H, alkyl,aryl or aralkyl, and a pharmaceutically acceptable salt thereof.
 3. Themethod of claim 2, wherein said apoptotic signal is induced bycontacting said cell with a compound selected from the group consistingof cytarabine, fludarabine, 5-fluoro-2′-deoxyuiridine, gemcitabine,methotrexate, bleomycin, cisplatin, cyclophosphamide, adriamycin(doxorubicin), mitoxantrone, camptothecin, topotecan, colcemid,colchicine, paclitaxel, vinblastine, vincristine, tamoxifen,finasteride, taxotere and mitomycin C.
 4. The method of claim 2, whereinsaid apoptotic signal is induced by contacting said cell withApo2L/TRAIL.